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How does high-fidelity of DNA replication depend on the formation of hydrogen bonds?

How does high-fidelity of DNA replication depend on the formation of hydrogen bonds?


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Replication has an error rate of less than 1 in 100 million. DNA polymerase forms H-bond with the H-bond acceptor atoms in the minor groove. <-- enhance fidelity here?

Binding of the triphosphate group to the active site of DNA polymerase triggers a conformational change. Changing a conserved Tyr residue increases the error rate by 40 fold.

I don't quite understand the above two statements. Can anyone explain in detail to me? Thanks!


DNA polymerase must catalyse the addition of 4 different nucleotides to the growing strand. This means that it cannot directly determine which base to incorporate at a specific point (how would it 'know' which base to incorporate and how it would it change its specificity for different bases). This means that the specificity for which base pair to incorporate is dependent on the template DNA strand.

Correct Watson-Crick base pairing (that is, hydrogen bonding) between the template strand and the nucleotide to be incorporated triggers the closing of the finger domain of DNAP around the primer-template junction and positions the latter in the optimal position for catalysis (with the $ce{alpha-PO_4}$ of the incoming nucleotide near the $ce{3'-OH}$ of the primer for a nucleophilic attack catalysed by two $ce{Mg^{2+}}$ ions). This is where the conserved tyrosine residue you mentioned comes into play. An incorrectly paired nucleotide will not trigger this conformational change and will not be positioned optimally, thus catalysis is less likely.

Furthermore, the DNA polymerase makes contacts with the minor groove of the primer-template junction through hydrogen bonds. This interaction is not base-specific (all Watson-Crick base pairs have he same pattern of hydrogen-bond acceptors in the minor groove) but only occurs when the correct nucleotide is incorporated, thus stabilising the complex.

Finally, discrimination between ribonucleotides and deoxyribonucleotides is done by steric exclusion of the $ce{2'-OH}$ by amino acid residues in the binding pocket.

These factors can be thought of as kinetic proofreading as they simply slow down the reaction rate and provide time for an incorrectly paired nucleotide to dissociate. However, they can still be incorporated and many DNA polymerases have a $ce{3'->5'}$ proofreading exonuclease that can remove incorrectly paired nucleotides. This proofreading is again mediated by interactions between the DNAP and the primer-template junction (ie hydrogen bonding with the minor groove). A weakened interaction due to an incorrectly paired base reduces the affinity between DNA and the catalytic site and increases the affinity between DNA and the proofreading site (because it has a preference for cleaving ssDNA from the 3' end).


High-fidelity DNA polymerases have several safeguards to protect against both making and propagating mistakes while copying DNA.

Such enzymes have a significant binding preference for the correct versus the incorrect nucleoside triphosphate during polymerization.

If an incorrect nucleotide does bind in the polymerase active site, incorporation is slowed due to the sub-optimal architecture of the active site complex. This lag time increases the opportunity for the incorrect nucleotide to dissociate before polymerase progression, thereby allowing the process to start again, with a correct nucleoside triphosphate (1,2).

If an incorrect nucleotide is inserted, proofreading DNA polymerases have an extra line of defense

The perturbation caused by the mispaired bases is detected, and the polymerase moves the 3' end of the growing DNA chain into a proofreading 3'→5' exonuclease domain. There, the incorrect nucleotide is removed by the 3'→5' exonuclease activity, whereupon the chain is moved back into the polymerase domain, where polymerization can continue.

From: New England biolabs:

https://www.neb.com/tools-and-resources/feature-articles/polymerase-fidelity-what-is-it-and-what-does-it-mean-for-your-pcr


Both the sentence can be understood from the below explanation.

In a newly synthesizing DNA strand, the DNA polymerase forms extensive hydrogen bonds with the newly added base everytime a new nucleotide is added. (First sentence) Polymerase also forms bonds with the DNA backbone of phosphate and sugar. (Second sentence)

The first sentence talks of how polymerase will form bonds via the minor groove of the newly synthesized DNA. Second sentence talks about the changing of an amino acid from the DNA polymerase (which is important for forming bonds with DNA backbone). This changing of amino acid will cause error in the nucleotide added (as the Polymerase is not positioned properly which you will understand in the following explaination)

These bonds of polymerase and bases along with the bonds between the DNA backbone and polymerase position the DNA polymerase.

When a newly added nucleotide is correctly base paired, the structure of the paired bases will be such that the bonds formed between polymerase and base will allow DNA polymerase to be positioned for the addition of next new nucleotide. If a incorrect nucleotide was added the DNA polymerase positions such that it's exonuclease site is at a position to catalyse exonuclease activity.

Thus, the hydrogen bonds between the base and Polymerase is needed for correct positioning of polymerase to either allow the next nucleotide to be added (if correct base was added) or allow exonuclease activity of Polymerase (if an incorrect base was added). This will help in proofreading and thus provide high fidelity.


6 Process and Types Involved in Replication of DNA (With Diagram)

It has now been over 30 years since J. D. Watson, F. H. C. Crick, and M. H. F. Wilkins established the double-stranded, helical nature of the DNA molecule and suggested how DNA serves in its own replication.

According to their original model for DNA replication, the two polynucleotide chains of the “parent” double helix separate and each serves as a “template” for the synthesis of a new, complementary polynucleotide chain.

During strand separation, the nitrogen bases of each original strand are exposed and establish sites for the association of free nucleo­tides.

These nucleotides are then enzymatically linked together to form a new complementary strand. Because deoxyadenylic acid (dAMP) can form hydrogen bonds only with thymine of the template strand (and dGMP can bond only to cytosine, dCMP only to guanine, and dTMP only to ade­nine), the newly synthesized strand will be identical to the original complementary strand.

As a result, two new double helices are formed, each consisting of one polynucleotide strand from the parent double helix and a newly synthesized polynucleotide strand. Be­cause each of the two double helices conserves only one of the parent polynucleotide strands, the process is said to be semiconservative.

Replication as a “Semiconservative” Process:

Although semiconservative replication of DNA was predicted by the original Watson-Crick model, it was not verified until the classic studies of M. S. Meselson and F. W. Stahl. At the time of their experiments, two other modes of replication were deemed equally feasi­ble (Fig. 21-1):

(1) Conservative replication, in which both strands of the parent double helix would be con­served and the new DNA molecule would consist of two newly synthesized strands and

(2) Dispersive replication, in which replication would involve fragmentation of the parent double helix and the in­terspersing of pieces of the parent strands with newly synthesized pieces, thereby forming the two new dou­ble helices.

Meselson and Stahl verified the semiconservative nature of DNA replication in a series of elegant exper­iments using isotopically labeled DNA and a form of isopycnic density gradient centrifugation (see Chap­ter 12). They cultured Escherichia coli cells in a me­dium in which the nitrogen was 15 N (a “heavy” isotope of nitrogen, but not a radioisotope) instead of the com­monly occurring and lighter 14 N.

In time, the purines and pyrimidines of DNA in new cells contained 15 N (where 14 N normally occurs) and thus the DNA mole­cules were denser. DNA in which the nitrogen atoms are 15 N can be distinguished from DNA containing 14 N because during isopycnic centrifugation, the two dif­ferent DNAs band at different density positions in the centrifuge tube (Fig. 21-2).

Meselson and Stahl centrifuged DNA isolated from the cells for 2-3 days at very high rotational speeds in centrifuge tubes initially containing a uniform solu­tion of CsCl. During centrifugation, density gradients were automatically formed in the tubes as a result of the equilibrium that was established between the sedi­mentation of CsCl toward the bottom of the tube and diffusion of the salt toward the top of the tube. This form of centrifugation, called equilibrium isopycnic centrifugation,

Depend­ing on its content of 15 N and 14 N, the DNA bands at a specific position in the density gradient. Because the DNA synthesized by cells grown in 15 N would be denser than 14 N-containing DNA it would band fur­ther down the tube (Fig. 21-2).

Cells grown for some time in the presence of 15 N- medium were washed free of the medium and trans­ferred to 14 N-containing medium and allowed to con­tinue to grow for specific lengths of time (i.e., for various numbers of generation times). DNA isolated from cells grown for one generation of time in the 14 N medium had a density intermediate to that of the DNA from cells grown only in 15 N-containing medium (identified as generation 0 in Fig. 21-3) and that of DNA from cells grown only in 14 N-containing medium (the controls of Fig. 21-3).

Such a result immediately ruled out the possibility that DNA replication was con­servative, as conservative replication would have yielded two DNA bands in the density gradient for generation 1 (i.e., F,) cells. The single band of inter­mediate density (identified as “hybrid” DNA in Fig. 21-3) consisted of DNA molecules in which one strand contained 15 N and the other contained 14 N.

When the incubation in 14 N medium was carried out for two gen­erations of time (i.e., generation 2), two DNA bands were formed—one at the same density position as the DNA from cells grown exclusively in 14 N medium (i.e., “light controls”) and one of intermediate density. Sub­sequent generations produced greater numbers of DNA molecules that banded at the “light” ( 14 N- containing DNA) position in the density gradient. These results are consistent only with the model of semiconservative replication.

Dispersive replication would have produced a single band for each genera­tion and the band would have been found at succes­sively lighter density positions in the gradient. Stud­ies using other prokaryotes as well as eukaryotes indicate that semiconservative replication of DNA is probably the universal mechanism.

Replication by Addition of Nucleotides in the 5′3′ Direction:

Each nucleotide of a DNA strand is joined to the next nucleotide by a phosphodiester bond that links the 3′ carbon of its deoxyribose to the 5′ carbon of the deoxyribose of the next nucleotide. At one end of the polynucleotide chain, there is a hydroxyl group attached to the 3′ carbon of the last nucleotide, and at the other end there is a phosphate group at­tached to the 5′ carbon.

The two chains of a double he­lix have opposite polarities and are said to be antiparallel, that is, each end of the double helix contains the 5′ end of one strand and the 3′ end of the other. Dur­ing replication, attachment of a nucleotide to a grow­ing strand always takes place at the terminal 3′ posi­tion of that strand. In other words, the forming polynucleotide chain “grows” from its 5′ end toward its 3′ end.

Unidirectional and Bidirectional Replication:

Replication starts at a point on the chromosome where the two parental strands begin to separate this point is called the origin. Addition of complementary nucleotides to form two new strands takes place along both parent strand templates starting from that point (Fig. 21-4).

In unidirectional replication, growth pro­ceeds along both strands in the same direction leading from the origin. Along one of the parental template strands, synthesis of the new complementary strand takes place by the continuous addition of nucleotides to the available 3′ end of the forming strand. The growing strand is called the leading strand or contin­uous strand. The 5′ end of this strand is located at the origin and its 3′ end at the moving replication fork (i.e., the progressing point of separation of the paren­tal strands).

The other polynucleotide strand being formed is called the lagging strand or discontinuous strand. The elongation of this strand takes place by a some­what modified mechanism. In contrast to the leading strand, the lagging strand has its 3′ position at the ori­gin and its 5′ position at the replication fork. If nucle­otides were sequentially added to the end of the lag­ging strand at the replication fork, then this strand’s growth would proceed in a 3’→5′ direction.

This does not occur. Instead, growth takes place by the synthe­sis of a number of short polynucleotide chains be­tween the replication fork and the origin. Each short chain is laid down in the direction 5′ to 3′ and these are later linked together and to the 5′ end of the lag­ging strand.

As a result, the overall direction of growth of the lagging strand is the same as that of the leading strand. The unusual growth pattern that char­acterizes the synthesis of the lagging strand explains why it is also referred to as the “discontinuous” strand.

In bidirectional replication (Fig. 21-5), two repli­cation forks are formed at the origin and these move away from the origin in both directions as the parental double helix is separated. The synthesis of the comple­mentary strands also occurs in both directions. Be­hind each fork there is a set of leading and lagging strands. As in the case of unidirectional replication, elongation of the two leading strands is continuous, whereas elongation of the two lagging strands is dis­continuous.

It is to be noted that regardless of whether replication is unidirectional or bidirectional, the addition of nucleotides always occurs in the direc­tion from 5′ to 3′, as new nucleotides are added to available 3′ ends of either the continuous strand or the discontinuous strand. Discontinuous synthesis of lagging strands was first demonstrated by R. Okazaki. Okazaki incubated E. coli cells in a medium containing 3 H-thymidine for very short periods of time (a pulse of only 15 seconds) and then examined the distribution of the radioisotope in newly synthesized DNA.

The radioisotope was found in a number of polynucleotides (1000-2000 nu­cleotides long), now referred to as Okazaki frag­ments (Figs. 21-4 and 21-5). When pulsed cells were transferred to unlabeled medium for varying lengths of time prior to analysis, the radioactive label was re­covered in much longer stretches of DNA. This is be­cause the Okazaki fragments produced during the short tritium pulse had been linked together and con­nected to the 5′ end of the lagging strand.

In eukaryotic cells, Okazaki fragments are usually smaller (about 100-200 nucleotides long). Bidirectional replication of DNA is the mechanism employed in all eukaryotic and most prokaryotic cells. Unidirectional replication is rare and appears to occur in only a limited number of prokaryotes.

Visualization of Replication in E. coli:

In 1963, J. Cairns developed a procedure employing a combination of microscopy and autoradiography that made it possible to visualize the replication of the chromosome of E. coli. Cairns plaped E. coli cells in a medium containing 3 H-thymidine for various periods of time so that the radioactive thymidine was incorpo­rated into the DNA as the chromosome was replicated in successive generations of cells.

Cells were removed from the medium after various periods of incubation and gently lysed to release the chromosome from the cell (the shear forces created by harsh lysis break the chromosome into small pieces). The chromosomes were then transferred to glass slides and coated with a photographic emulsion sensitive to the low-energy beta particles emitted by the 3 H-thymidine.

After ex­posing the emulsion to the beta rays, the emulsion was developed and examined by light microscopy. Wherever decay of labeled thymidine had occurred in a chromosome, the emulsion was exposed and created visible grains.

A chromosome not engaged in replication appeared as a circular structure formed from a close succession of exposure spots. Chromosomes “caught in the act” of replication gave rise to what are called theta struc­tures because they have the appearance of the Greek letter theta (i.e., 0) (Fig. 21-6). The theta structures reveal the positions of the replication forks in the cir­cular chromosome.

The Replicon and the Replication Sequence:

The sequence of events that takes place during DNA replication is best understood for prokaryotes and ap­pears to be as follows (Fig. 21-7). Parental strand sep­aration begins at a site called the origin which con­tains a special nucleotide sequence and directs the association of a number of proteins. ATP-dependent unwinding enzymes (also called helicases) promote separation of the two parental strands and establish replication forks that will progressively move away from the origin (Fig. 21-7a) the helicases separate the parental strand at about 1000 base pairs per sec­ond.

Behind the replication fork, the single DNA strands are prevented from rewinding about one an­other (or forming double-stranded hairpin loops in each single strand) by the actions of a set of proteins called helix-destabilizing proteins or single-strand binding proteins (i.e., “SSBs”) (Fig. 21-7b). The action of a helicase introduces a positive supercoil into the duplex DNA ahead of the replication fork. En­zymes called topoisomerases relax the supercoil by attaching to the transiently supercoiled duplex, nick­ing one of the strands, and rotating it through the un­broken strand. The nick is then resealed.

Prior to DNA synthesis beginning at the origin, short RNA polynucleotides are formed that are com­plementary to the DNA template. These stretches of RNA are called primers and are also laid down in the 5′ to 3′ direction. DNA nucleotides are then added one at a time to the free 3′ ends of the RNA primers. Be­cause growth of the lagging strand is discontinuous, several RNA primers and Okazaki fragments are formed. Note that an RNA primer must be formed for each Okazaki fragment to be laid down (Fig. 21-7c). The enzymes required for the synthesis of the RNA primers are a special class of RNA polymerases called RNA primases.

Elongation of the leading strand and synthesis of the Okazaki fragments are catalyzed by an enzyme called DNA polymerase III. The substrates of DNA polymerase III are the deoxynucleoside triphosphates (e.g., dATP, dGTP, dCTP, and dTTp). Addition of a nu­cleotide to the available 3′ position of the continuously growing leading strand or an Okazaki fragment of the lagging strand involves removal of pyrophosphate to yield a deoxynucleoside monophosphate (e.g., dAMP, dGMP, dCMP, and dTMP).

On completion of the Oka­zaki fragments, the RNA primers are excised by DNA polymerase I, which then fills the resulting gaps with DNA (Fig. 21-7d). After DNA polymerase I adds the final deoxyribonucleotide in the gap left by the ex­cised primer, the enzyme DNA ligase forms the phosphodiester bond that links the free 3′ end of the primer replacement to the 5′ end of the Okazaki frag­ment (Fig. 21-7f).

DNA Polymerases and “Processivity”:

In general, three different DNA polymerase enzymes are found in cells. In prokaryotes, these are called DNA poly­merase I, DNA polymerase II, and DNA polymerase III. As noted above, DNA polymerase I excises the RNA primers and fills the gaps with DNA, whereas DNA polymerase III adds nucleotides to the growing leading strand and to the 3′ ends of the RNA primers.

The function of DNA polymerase II remains unknown. In eukaryotic cells, the DNA polymerases are DNA polymerase a, DNA polymerase II, and DNA poly­merase 7 their functions are compared with the pro- karyotic enzymes in Table 21-1. The rapidity and effi­ciency with which a DNA polymerase extends a growing chain is referred to as processivity. For exam­ple, the processivity of DNA polymerase III acting on the 3′ end of the leading strand is very high because the enzyme remains associated with the growing end of the chain and the template strand.

During unidirectional replication, the replication fork fully circles the chromosome and the resulting DNA molecules are separated. For prokaryotes in which replication is bidirectional, the replication forks proceed around the chromosome until they meet. In eukaryotic chromosomes, where there are many repli­cating units or replicons, all replicons are linked to­gether before the chromatids can be -separated. The replicon consists of that segment of a chromosome that includes an origin and two termination points (i.e., points where replication ends).

N. K. Sinha and A. Kornberg have suggested that the DNA polymerases, RNA primases, and helicases may be associated with each other to form a multienzyme complex, the replisome that car­ries out the synthesis of the leading and lagging strands in a coordinated fashion (Fig. 21-8). Such a complex would be highly processive and assure rapid replication of the DNA.

High Fidelity of Replication:

Despite the complexity of the process and the rapidity with which it proceeds, very few errors are made dur­ing DNA replication. For example, it is estimated that for every error that occurs, 10 9 base pairs are repli­cated faithfully. The high fidelity of DNA replication is attributable in part to the special properties of the DNA polymerases.

The DNA polymerases will add a nucleotide to the available 3′-OH end of a growing DNA strand (or RNA primer) only if the prior nucleo­tide is properly base-paired with the template nucleo­tide. If a mismatched nucleotide is present, growth of the strand is transiently halted while a segment of the strand containing the error is excised. With a cor­rected 3′ end reestablished, elongation by the DNA polymerase is resumed.

Much remains to be learned about the enzymes that catalyze the reactions of replication. So far, the major obstacle to such studies has been the difficulty of iso­lating and purifying the enzymes, either individually or in complexes. This is due in part to the fact that some of them may be associated with membranes. In prokaryotic cells, the replication forks are bound to the plasma membrane.

About two dozen different pro­teins have been shown to be involved with the replica­tion of DNA in E. coli cells. Several of the E. coli pro­teins are involved with prepriming reactions, that is, the reactions that occur before the formation of RNA primers.


14.1 | Historical Basis of Modern Understanding

By the end of this section, you will be able to:

  • Explain transformation of DNA.
  • Describe the key experiments that helped identify that DNA is the genetic material.
  • State and explain Chargaff’s rules

Modern understandings of DNA have evolved from the discovery of nucleic acids to the development of the double-helix model. In the 1860s, Friedrich Miescher (Figure 14.2), a physician by profession, was the first person to isolate phosphate- rich chemicals from white blood cells or leukocytes. He named these chemicals (which would eventually be known as RNA and DNA) nuclein because they were isolated from the nuclei of the cells.

Figure 14.2 Friedrich Miescher (1844–1895) discovered nucleic acids.

A half century later, British bacteriologist Frederick Griffith was perhaps the first person to show that hereditary information could be transferred from one cell to another “horizontally,” rather than by descent. In 1928, he reported the first demonstration of bacterial transformation, a process in which external DNA is taken up by a cell, thereby changing morphology and physiology. He was working with Streptococcus pneumoniae, the bacterium that causes pneumonia. Griffith worked with two strains, rough (R) and smooth (S). The R strain is non-pathogenic (does not cause disease) and is called rough because its outer surface is a cell wall and lacks a capsule as a result, the cell surface appears uneven under the microscope. The S strain is pathogenic (disease-causing) and has a capsule outside its cell wall. As a result, it has a smooth appearance under the microscope. Griffith injected the live R strain into mice and they survived. In another experiment, when he injected mice with the heat-killed S strain, they also survived. In a third set of experiments, a mixture of live R strain and heat-killed S strain were injected into mice, and—to his surprise—the mice died. Upon isolating the live bacteria from the dead mouse, only the S strain of bacteria was recovered. When this isolated S strain was injected into fresh mice, the mice died. Griffith concluded that something had passed from the heat-killed S strain into the live R strain and transformed it into the pathogenic S strain, and he called this the transforming principle (Figure 11.3). These experiments are now famously known as Griffith’s transformation experiments.

Figure 14.3 Two strains of S.pneumoniae were used in Griffith’s transformation experiments. The R strain is non- pathogenic. The S strain is pathogenic and causes death. When Griffith injected a mouse with the heat-killed S strain and a live R strain, the mouse died. The S strain was recovered from the dead mouse. Thus, Griffith concluded that something had passed from the heat-killed S strain to the R strain, transforming the R strain into S strain in the process. (credit “living mouse”: modification of work by NIH credit “dead mouse”: modification of work by Sarah Marriage)

Scientists Oswald Avery, Colin MacLeod, and Maclyn McCarty (1944) were interested in exploring this transforming principle further. They isolated the S strain from the dead mice and isolated the proteins and nucleic acids, namely RNA and DNA, as these were possible candidates for the molecule of heredity. They conducted a systematic elimination study. They used enzymes that specifically degraded each component and then used each mixture separately to transform the R strain. They found that when DNA was degraded, the resulting mixture was no longer able to transform the bacteria, whereas all of the other combinations were able to transform the bacteria. This led them to conclude that DNA was the transforming principle.

Experiments conducted by Martha Chase and Alfred Hershey in 1952 provided confirmatory evidence that DNA was the genetic material and not proteins. Chase and Hershey were studying a bacteriophage, which is a virus that infects bacteria. Viruses typically have a simple structure: a protein coat, called the capsid, and a nucleic acid core that contains the genetic material, either DNA or RNA. The bacteriophage infects the host bacterial cell by attaching to its surface, and then it injects its nucleic acids inside the cell. The phage DNA makes multiple copies of itself using the host machinery, and eventually the host cell bursts, releasing a large number of bacteriophages. Hershey and Chase labeled one batch of phage with radioactive sulfur, 35 S, to label the protein coat. Another batch of phage were labeled with radioactive phosphorus, 32 P. Because phosphorous is found in DNA, but not protein, the DNA and not the protein would be tagged with radioactive phosphorus.

Each batch of phage was allowed to infect the cells separately. After infection, the phage bacterial suspension was put in a blender, which caused the phage coat to be detached from the host cell. The phage and bacterial suspension was spun down in a centrifuge. The heavier bacterial cells settled down and formed a pellet, whereas the lighter phage particles stayed in the supernatant (the liquid above the pellet). In the tube that contained phage labeled with 35 S, the supernatant contained the radioactively labeled phage, whereas no radioactivity was detected in the pellet. In the tube that contained the phage labeled with 32 P, the radioactivity was detected in the pellet that contained the heavier bacterial cells, and no radioactivity was detected in the supernatant. Hershey and Chase concluded that it was the phage DNA that was injected into the cell and carried information to produce more phage particles, thus providing evidence that DNA was the genetic material and not proteins (Figure 14.4).

Figure 14.4 In Hershey and Chase’s experiments, bacteria were infected with phage radiolabeled with either 35S, which labels protein, or 32P, which labels DNA. Only 32P entered the bacterial cells, indicating that DNA is the genetic material.

Around this same time, Austrian biochemist Erwin Chargaff examined the content of DNA in different species and found that the amounts of adenine, thymine, guanine, and cytosine were not found in equal quantities, and that it varied from species to species, but not between individuals of the same species. He found that the amount of adenine equals the amount of thymine, and the amount of cytosine equals the amount of guanine, or A = T and G = C. These are also known as Chargaff’s rules. This finding proved immensely useful when Watson and Crick were getting ready to propose their DNA double helix model, discussed in Chapter 5.


Directionality

These strands have two designated ends called 5&rsquo and 3&rsquo (you can read that as 5 prime end and 3 prime end). These numbers indicate end-to-end chemical orientation. The numbers 5 and 3 represent the fifth and third carbon atom of the sugar ring respectively. 5&rsquo is the end, which joins a phosphate group that attaches to another nucleotide. 3&rsquo end is important as during replication the new nucleotide is added to this end.

In terms of direction, if one strand is 5&rsquo to 3&rsquo while reading from left to right, the other strand will be 3&rsquo to 5&rsquo. Simply put, the strands run in opposite directions. This orientation is kept for easy binding between nucleotides of the opposite strands.

The chemical structure of a four base pair fragment of a DNA double helix. (Photo Credit : Thomas Shafee / Wikimedia Commons)


How does high-fidelity of DNA replication depend on the formation of hydrogen bonds? - Biology

We have characterized the role of Watson-Crick hydrogen bonding in the 3′-terminal base pair on the 3′-5′ exonuclease activity of the human mitochondrial DNA polymerase. Nonpolar nucleoside analogs of thymidine (dF) and deoxyadenosine (dQ) were used to eliminate hydrogen bonds while maintaining base pair size and shape. Exonuclease reactions were examined using pre-steady state kinetic methods. The time dependence of removal of natural nucleotides from the primer terminus paired opposite the nonpolar analogs dF and dQ were best fit to a double exponential function. The double exponential kinetics as well as the rates of excision (3–6 s –1 fast phase, 0.16–0.3 s –1 slow phase) are comparable with those observed during mismatch removal of natural nucleotides even when the analog was involved in a sterically correct base pair. Additionally, incorporation of the next correct base beyond a nonpolar analog was slow (0.04–0.22 s –1 ), so that more than 95% of terminal base pairs were removed rather than extended. The polymerase responds to all 3′-terminal base pairs containing a nonpolar analog as if it were a mismatch regardless of the identity of the paired base, and kinetic partitioning between polymerase and exonuclease sites failed to discriminate between correct and incorrect base pairs. Thus, sterics alone are insufficient, whereas hydrogen bond formation is essential for proper proofreading selectivity by the mitochondrial polymerase. The enzyme may use the alignment and prevention of fraying provided by proper hydrogen bonding and minor groove hydrogen bonding interactions as critical criteria for correct base pair recognition.


Hydrogen bonding revisited: Geometric selection as a principal determinant of DNA replication𠂟idelity

That hydrogen bonds play a central role in forming Watson𠄼rick (W𠄼) A⋅T and G𢱜 base pairs is a fundamental paradigm dating from the discovery of the structure of DNA in 1953. In addition to interstrand H bonding, intrastrand base-stacking and interstrand cross-stacking interactions are important in maintaining the bases in a stacked structure along the length of the DNA backbone. In general, H bonds between W𠄼 base pairs are viewed as “informational,” whereas the base-stacking interactions are regarded as “noninformational,” merely stabilizing the double helix. Consequently, it is a common perception that the H bonds pairing A with T and G with C are primarily responsible for the ability of DNA polymerases to synthesize DNA with high fidelity.

Difluorotoluene, a nonpolar isosteric analog of thymine (T), contains fluorine atoms in place of oxygens on the pyrimidine ring and thus cannot form H bonds with A (1). Nevertheless, A𢱟 base pairs are formed almost as well as A⋅T pairs by Escherichia coli proofreading-defective DNA polymerase I (KF exo − ), as Moran et al. (2) report in this issue of the Proceedings [the chemical structures of F and T and space-filling models of each are shown in Moran et al. (2), figure 1]. The observation that KF exo − fails to discriminate strongly against A𢱟 pairs applies when F is present either as a template base on DNA (3) or as a dFTP substrate (2). The apparently inescapable conclusion is that H bonds are not absolutely required for polymerase to form W𠄼 base pairs selectively.

These results provide an impetus to reconsider what role H bonds actually play in stabilizing DNA and enhancing DNA polymerase fidelity. Mismatched base pairs in a duplex DNA oligomer do cause marked reductions in DNA melting temperatures (4). The loss of H bonds upon replacement of T with F has this type of destabilizing effect (2). However, the notion that H bonds alone keep the two strands of a DNA double helix together, which is found in many textbooks, seems inadequate. When one considers that duplex alternating copolymers poly d(A,T) or poly d(G,C) have melting temperatures in aqueous solution that differ substantially from their respective homopolymer counterparts poly dA⋅poly dT or poly dG⋅poly dC, it becomes clear that base-stacking interactions have an important, perhaps dominant, sequence-dependent effect on duplex stability.

Furthermore, the free-energy differences (ΔΔG 0 ) between matched and mismatched base pairs deduced from melting data are in a range of about 0.2𠄴.0 kcal/mol (4𠄶), depending on the identity of the mispair, the surrounding sequence context, and its location near the center or at the DNA terminus. These ΔΔG 0 values, as measured in solution, are insufficient to account for the high nucleotide insertion fidelities of virtually all polymerases, including those that seem to be especially 𠇎rror prone” such as eukaryotic Pol β (7) or HIV-1 reverse transcriptase (8�). For example, ΔΔG 0 𢒃.7 kcal/mol measured for the natural base pairs A⋅T versus A𢱜 (6) should result in an A𢱜 misinsertion frequency of about 2 × 10 𢄣 . However, dAMP𢱜 and dCMP𢱚 misinsertion frequencies are typically about one to two orders of magnitude lower than that (11).

The recognition that base-pairing free-energy differences are too small to completely account for polymerase insertion selectivities prompted H. Echols and me to propose a “geometric selection” mechanism as a key component of insertion specificity (12, 13). The idea is that geometrical and electrostatic properties of the polymerase active site are likely to have a profound influence on nucleotide-insertion specificities. This influence would strongly favor insertion of bases having an optimal geometry, such that the C1′ distances and bond angles most closely approximate those of the Watson𠄼rick base pairs. For example, G⋅T, G𢱚, and C𢱚 mispairs have markedly different bond angles than A⋅T and G𢱜 pairs (Fig. ​ (Fig.1) 1 ) (14).

Geometric properties of Watson𠄼rick and mismatched base pairs. This figure is based on x-ray crystallography of duplex B-DNA oligonucleotides. The striking geometric identity of the Watson𠄼rick A⋅T and G𢱜 base pairs is not matched by the A𢱜 protonated wobble and G⋅T wobble base mispairs or by the G(anti)𢱚(syn) base mispair. [Reproduced with permission from ref. 14 (copyright 1987, Springer, Heidelberg).]

The observation by Moran et al. (2) that insertion of the base analog F opposite A is reduced by only 40-fold compared with its isosteric parent compound T opposite A suggests that the geometrical alignment of the substrate and template bases is a major determinant of polymerase fidelity. This result can be compared with earlier studies using the base analog 2-aminopurine (2AP), which forms 2AP⋅T base pairs with two H bonds in a proper W𠄼 geometry and is reduced by 7-fold compared with A⋅T (15, 16). Thus, the absence of H bonds in the incorporation of F opposite A decreases selectivity only about 6-fold relative to incorporation of 2AP opposite T. Considering that mispairs assuming non-W𠄼 geometries such as G⋅T (wobble), A𢱜 (protonated wobble), G𢱚 (antisyn) (Fig. ​ (Fig.1) 1 ) are misinserted with frequencies on the order of 10 𢄣 � 𢄦 (11), geometric constraints imposed at the polymerase active site may improve selectivities by perhaps three orders of magnitude or more.

There are at least three possible check points for proper geometric alignment during base insertion by polymerases: initial dNTP binding (16, 17), postbinding selection for the correct geometry (12, 18) by an induced-fit mechanism (19�), and the chemical step of phosphodiester formation. Previous data suggest there are significant differences in the extent to which different polymerases use each of the check points. We have suggested that the remarkable base-insertion fidelity of DNA polymerases derives from the sequential application of each check point to provide exquisite sensitivity to Watson𠄼rick geometry at the transition state for phosphodiester formation (13).

Taking the geometrical constraints imposed by the polymerase active site into consideration in conjunction with the active site electrostatic environment, it may be possible to relate the polymerase-insertion selectivity to solution free-energy differences between matched and mismatched base pairs. Measurements of ΔG 0 = ΔH 0 − TΔS 0 indicate relatively small differences between right and wrong base pairs at 37ଌ ΔΔG 0 is in a range of 0.2𠄴 kcal/mol, as mentioned above. The differences are small because ΔS 0 correlates with ΔH 0 (5, 22), a phenomenon called enthalpy𠄾ntropy compensation, which is observed in aqueous solution (23). That is, it takes more energy to melt highly stable, rigidly constrained base pairs than it does to melt less stable, weakly constrained base pairs. However, rigid base pairs having fewer degrees of freedom in the double helix will gain more degrees of freedom upon melting, whereas the opposite is true for less stable base pairs. As long as entropy and enthalpy changes are proportional, ΔΔH 0 is reduced by TΔΔS 0 , resulting in a small ΔΔG 0 (5).

How might polymerases increase free-energy differences to achieve high discrimination? Perhaps the geometric constraints imposed on the substrate and template bases in the polymerase active cleft can suppress ΔΔS enough to bring ΔΔG much closer in magnitude to ΔΔH. Typical values of ΔΔH 0 measured in aqueous solution are, by themselves, almost large enough to accommodate polymerase-insertion fidelities (5). Then, to the extent that dNTP and template bases confront each other in a lower dielectric medium that acts to partially exclude water, ΔΔH values may be even larger than in water (24). Thus, a polymerase active site that snugly accommodates correct base pairs by geometric selection and also reduces water in the vicinity of the base pair may amplify base pair free-energy differences by reducing entropy differences and increasing enthalpy differences by amounts sufficient to account for nucleotide-insertion fidelity.

In addition to discrimination during nucleotide insertion, fidelity in DNA replication is often enhanced by an associated exonuclease activity. Proofreading exonucleases increase fidelity by approximately 40- to 200-fold (25), displaying significantly less selectivity than polymerases. It is generally believed that exonuclease relies on the “melting capacity” of the 3′ terminus to distinguish between correct and incorrect insertions (26, 27). It is reasoned that polymerization and proofreading are competing reactions at a primer-3′ terminus, requiring an annealed or melted terminus, respectively (16, 17). Discrimination arises because the 3′ terminus is more likely to be annealed following correct insertions, favoring polymerization, but much more likely to be melted out following incorrect insertions, favoring excision (16, 28). It is tempting to ask if a geometric selection mechanism might be occurring in the exonuclease active site, enhancing the excision of non-Watson𠄼rick base pairs beyond what would be expected based solely on the relative stabilities of base pairs in solution.

Qualitatively, differences in base pair stabilities appear to be sufficient. Evidence comes from presteady state kinetics measurements on the excision of 2AP paired opposite T, C, A, and G using bacteriophage T4 DNA polymerase (29). The rate of excision of 2AP from a primer-3′ terminus is inversely correlated with the melting temperature of 2AP⋅N base pairs imbedded in an oligomer DNA duplex. For example, when present in the same sequence context, a “stable” 2AP⋅T Watson𠄼rick base pair is hydrolyzed much more slowly than an unstable 2AP𢱜 wobble mispair. However, a terminal 2AP⋅T in a A-T rich environment is hydrolyzed more rapidly than 2AP𢱜 in a G-C rich environment. Thus, exonuclease specificity appears to be more strongly tied to DNA stability than to terminal base pair geometry.

Nevertheless, it would be of great interest to use proofreading-proficient polymerases to measure the excision of difluorotoluene. Based on thermal denaturation measurements, Moran et al. (2) demonstrate that F pairs poorly with all of the natural bases. Significantly, the magnitude of the free energy difference between F𢱚 and T𢱚 base pairs is 3.6 kcal/mol, similar to that found for C𢱚 versus T𢱚 base pairs (6). If proofreading activities are governed primarily by primer stability and not geometric selection, then one would expect F to be excised much more rapidly than T opposite A, and, furthermore, the rate of excision of F might be the same whatever natural bases with which it paired.

The surface has barely been scratched in terms of understanding the interactions between polymerases and DNA that determine replication fidelity. The magnitude and location of mutations depend on a complex interplay between polymerases, proofreading exonucleases, processivity factors and the properties of the DNA primer-template sequences. Although models have been proposed to include polymerase steady-state kinetic parameters along with base stacking and sequence context to explain fidelity (11), precise molecular mechanisms governing mutagenic hot and cold spots remain obscure. Different polymerases copying the same primer-template DNA can exhibit markedly different mutation frequencies and spectra. The ability to separate the effects of H bonding from base stacking holds the promise of new progress in these directions. The future use of the difluorotoluene T analog along with an anticipated group of other non-H bonding base analogs should enable a precise determination of the effects of nearest-neighbor base stacking on misinsertion frequencies and proofreading efficiencies, and shed light on how different polymerase and exonuclease active sites sense the presence of nearby primer and template bases.

I acknowledge the fundamental contribution of Hatch Echols in recognizing the importance of geometric selection in the determination of polymerase fidelity, and I thank D. Kuchnir Fygenson, John Petruska, Ken Breslauer, and Sharon Wald Krauss for their insightful, intellectual contributions and generous advice. This work was supported by the National Institutes of Health (GM21422) and by the Hedco Molecular Biology Laboratory at the University of Southern California.


B. DNA Polymerases Catalyze Replication

The first of these enzymes was discovered in E. coli by Arthur Kornberg, for which he received the 1959 Nobel Prize in Chemistry. Thomas Kornberg, one of Arthur&rsquos sons later found two more of DNA polymerases! All DNA polymerases require a template strand against which to synthesize a new complementary strand. They all grow new DNA by adding to the 3&rsquo end of the growing DNA chain in successive condensation reactions. And finally, all DNA polymerases also have the odd property that they can only add to a pre-existing strand of nucleic acid, raising the question of where the &lsquopreexisting&rsquo strand comes from! DNA polymerases catalyze the formation of a phosphodiester linkage between the end of a growing strand and the incoming nucleotide complementary to the template strand. The energy for the formation of the phosphodiester linkage comes in part from the hydrolysis of two phosphates (pyrophosphate) from the incoming nucleotide during the reaction. While replication requires the participation of many nuclear proteins in both prokaryotes and eukaryotes, DNA polymerases perform the basic steps of replication, as shown in the illustration below.

Although DNA polymerases replicate DNA with high fidelity with as few as one error per 107 nucleotides, mistakes do occur. The proofreading ability of some DNA polymerases corrects many of these mistakes. The polymerase can sense a mismatched base pair, slow down and then catalyze repeated hydrolyses of nucleotides until it reaches the mismatched base pair. This basic proofreading by DNA polymerase is shown below.

After mismatch repair, DNA polymerase resumes forward movement. Of course, not all mistakes are caught by this or other repair mechanisms (see DNA Repair, below). Mutations in the eukaryotic germ line cells that elude correction can cause genetic diseases. However, most are the mutations that fuel evolution. Without mutations in germ line cells (egg and sperm), there would be no mutations and no evolution, and without evolution, life itself would have reached a quick dead end! Other replication mistakes can generate mutations somatic cells. If these somatic mutations escape correction, they can have serious consequences, including the generation of tumors and cancers.


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The diagram must show four nucleotides shown with two on each side showing phosphate-sugar backbones and nitrogen base pairs bonded between them.

Award [1] for each of the following clearly drawn and correctly labelled.
phosphate – shown connected to deoxyribose
deoxyribose – shown connected to phosphate
(nitrogenous) bases – shown bonded to deoxyribose
base pairs – shown with labels adenine/A bonded to thymine/T and cytosine/C bonded to guanine/G
hydrogen bonds – shown connecting bases
covalent bonds – shown connecting deoxyribose to phosphates
nucleotide – clearly identified by the candidate
Award [4 max] if diagram is not shown double stranded.

DNA samples are taken from crime scene, suspects and victims
polymerase chain reaction/PCR used to increase the amount of DNA
restriction enzymes used to cut DNA
electrophoresis involves electric field/placing sample between electrodes
used to separate DNA fragments according to size
creating DNA profiles/unique patterns of bands
comparison is made between the patterns
criminals/victims can be identified in this way
DNA is (quite) stable / DNA can be processed long after the crime

DNA codes for a specific sequence of amino acids/polypeptide
the DNA code for one polypeptide is a gene
DNA is transcribed into mRNA
mRNA moves to a ribosome
where mRNA is translated into a polypeptide
originally it was thought that one gene always codes for one polypeptide
some genes do not code for a polypeptide
some genes code for transfer RNA/tRNA/ribosomal RNA/rRNA
some sections of DNA code for regulators that are not polypeptides
antibody production does not follow this pattern (of simple transcription-translation) (allow other examples)
change in the gene/mutation will affect the primary structure of the polypeptide


Lesson Explainer: DNA Replication Biology

In this explainer, we will learn how to describe the process of semiconservative DNA replication, including the role of different enzymes, and recall how errors made during DNA replication can be corrected.

One of the key characteristics of any living organism is its ability to grow and reproduce. Both of these processes, at the cellular level, involve simple cell division, or the splitting of one cell into two. We know that every living cell carries genetic material in the form of DNA (deoxyribonucleic acid). The genetic material controls every characteristic of a living cell, from its size and appearance to the functions it performs.

When one cell divides into two, therefore, each new cell must contain a copy of the DNA in its nucleus for it to be able to function properly. For example, when a liver cell divides into two new liver cells, each new cell must receive a copy of the original cell’s DNA, so that it can perform its role to support the natural functions of the liver.

Key Term: Deoxyribonucleic acid (DNA)

DNA is the molecule that carries the genetic instructions for life. It is composed of two strands of deoxynucleotides that coil around each other to form a double helix.

DNA replication is the process by which a dividing cell generates a copy of its DNA. As we know, in eukaryotes, a molecule of DNA resides in the nucleus and is made of two individual strands that coil around one another to form a “twisted ladder” shape called the double helix as we can see in Figure 1. The process of DNA replication takes place in the nucleus of the cell and is controlled by a set of enzymes, each of which performs a specific function. In this explainer, we will learn how DNA replication takes place and understand the roles played by each of the enzymes involved.

Key Term: Double Helix

A double helix is a “twisted ladder” shape, specifically the shape of a molecule of DNA.

Key Term: DNA Replication

DNA replication is the process by which two identical DNA molecules are produced from a single original DNA molecule.

Before we begin learning about DNA replication, let’s quickly go over the basic structure of a DNA molecule.

As we mentioned earlier, a molecule of DNA contains two strands coiled around one another. These two strands are called polynucleotide chains, and they are made of repeating smaller units called nucleotides. As we can see in Figure 1, each nucleotide has three components: a pentose sugar, a phosphate group, and a nitrogenous base. Each nucleotide links to the next through covalent bonds called phosphodiester bonds.

Key Term: Nucleotide

A nucleotide is a monomer of a nucleic acid polymer. Nucleotides consist of a pentose sugar, a phosphate group, and a nitrogenous base.

The two polynucleotide chains connect to one another through the pairing of the nitrogenous bases facing each other on the inside of the ladder. In DNA, there are four different types of nitrogenous bases: adenine (A), guanine (G), thymine (T), and cytosine (C).

When the nitrogenous bases on one strand pair with the nitrogenous bases on the opposite strand, they follow certain base-pairing rules. In a molecule of DNA, adenine can only bind to thymine on the opposite strand, and guanine can only bind to cytosine. This rule is called “complementary base-pairing” and is one of the defining features of DNA. Adenine binds to thymine through two hydrogen bonds, while guanine binds to cytosine through three hydrogen bonds, as shown in Figure 2.

Key Term: Complementary Base-Pairing

DNA bases can pair according to specific rules, where adenine (A) binds to thymine (T), while guanine (G) binds to cytosine (C). In RNA, uracil (U) is substituted for thymine (T). These rules of complementary base-pairing are critical for DNA replication and transcription.

Another important feature of DNA is its antiparallel nature. Each DNA strand has two ends: one end is called the

end, and the other end is called the

end. These two ends are named for the last carbon atoms of the nucleotide at each end. The two strands are antiparallel as one strand runs in the

direction, while the other runs in the

direction, as shown in Figure 3.

carbon atom of every nucleotide is bonded to a hydroxyl group (

end of a strand of DNA, therefore, ends with a hydroxyl group, as you can see in Figure 3. The

carbon atom of every nucleotide is bonded to a phosphate group. The

end of a strand of DNA, therefore, ends with a phosphate group, represented by the yellow circles in Figure 3.

A molecule of DNA carries genetic information in the form of a genetic code, formed by the sequence of nitrogenous bases. A type of RNA called messenger RNA or mRNA is synthesized according to the sequence of DNA. This sequence encodes the information needed for the synthesis of proteins, which then go on to control the cell’s functions and characteristics.

Key Term: Genetic Code

The genetic code is formed by the sequence of nitrogenous bases in a strand of messenger RNA (mRNA) molecule that is synthesized from the DNA and codes for the information needed for a cell to synthesize specific proteins.

So how is this genetic code read? When we read English, we always read from left to right. Similarly, when a cell “reads” the sequence of nitrogenous bases to interpret the genetic code, it is read from the

end of the DNA strand to the

In 1953, when James Watson and Francis Crick proposed the double helix model of DNA that we are familiar with today, they also made an interesting observation about DNA replication. Their model was based on the base pair specificity of the two strands in a DNA molecule. They realized that the two strands carry complementary versions of the same sequence. Because of this feature, both strands of DNA in a molecule could potentially be used as templates to synthesize complementary strands creating two new double helices with the same information! The sequence of nitrogenous bases on each strand is used as a guide for the sequence of new, complementary bases required to make up the new strand.

In eukaryotes, which are organisms with a well-organized nucleus, the DNA within the nucleus is present in the form of highly coiled, linear structures called chromosomes. Each chromosome contains one molecule of DNA, and within each molecule, replication begins at several different points.

In prokaryotes, on the other hand, the genetic material is present in the form of a single, circular molecule of DNA in the central area of the cell but is not surrounded by a nuclear membrane. In this case, replication begins at one point, called the origin of replication.

Now that we have had a quick recap of the structure of a molecule of DNA, let’s take a look at the mechanism of DNA replication. This process is controlled by three main enzymes. Let’s walk through the steps of this process and learn about each of the enzymes as we go along.

In order for the two strands of a DNA molecule to be used to make two new DNA molecules in the nucleus, the two strands must unwind and separate, making their nitrogenous bases accessible. We know that the strands are held together by hydrogen bonds between the nitrogenous bases of the two strands. In order for the strands to separate, these hydrogen bonds must be broken. This is accomplished by an enzyme called DNA helicase.

Key Term: DNA Helicase

DNA helicase is the enzyme responsible for separating or unwinding two complementary strands of DNA by breaking the hydrogen bonds between them, creating the replication fork in preparation for DNA replication.

Figure 4 shows how the strands of DNA separate through the action of DNA helicase. As the hydrogen bonds between the two strands are broken, a “replication fork” is formed, which is so named because the two unwinding strands of DNA have a forked appearance. The replication fork is the point at which the DNA molecule unwinds into two separate strands. You can picture the DNA helicase enzyme “unzipping” the molecule of DNA, beginning at the replication fork.

Key Term: Replication Fork

The replication fork is formed by the two separated or unwound strands of DNA in preparation for DNA replication.

Example 1: Understanding the Role of DNA Helicase

In the process of DNA replication, what is the primary role of DNA helicase?

  1. DNA helicase detects and repairs any errors that are made by incorrect base-pairing during DNA replication.
  2. DNA helicase breaks the hydrogen bonds between base pairs, separating the two strands of DNA.
  3. DNA helicase forms phosphodiester bonds between nucleotides to form a strand of DNA.
  4. DNA helicase adds nucleotides to a growing DNA chain, synthesizing a strand of DNA complementary to the template strand.
  5. DNA helicase joins the gaps in the backbone between newly formed DNA fragments.

Answer

When a cell undergoes division, its DNA replicates itself, so as to provide each new cell with a copy of DNA, which can control the cell’s characteristics and functions. We know that a single molecule of DNA is composed of two complementary polynucleotide strands. Each of these strands is made up of multiple individual units called nucleotides that form a sequence of nitrogenous bases. The order or sequence of nitrogenous bases along a strand of DNA encodes the “genetic information” that needs to be replicated when a cell divides.

When two strands of DNA bind together to form the familiar double helix shape, they do so by pairing their nitrogenous bases according to the rules of complementary base-pairing, in which adenine binds to thymine through two hydrogen bonds and guanine binds to cytosine through three hydrogen bonds.

The sequence of nitrogenous bases on each strand of DNA in the double helix acts as a template for the formation of a new strand of DNA. In order for this to happen, the two strands of DNA must unwind and separate, so that their nitrogenous bases are accessible. This function is carried out by the enzyme DNA helicase. DNA helicase breaks the hydrogen bonds between the nitrogenous bases on each strand, “unzipping” the DNA and separating the two strands.

Now that we have this information, let’s take a look through the options in the question. The one that best fits what we have learned is “DNA helicase breaks the hydrogen bonds between base pairs, separating the two strands of DNA,” and therefore, this is the correct option.

Once the strands have unwound, new strands of DNA that are complementary to each of the original strands can be synthesized. This is where an enzyme called DNA polymerase comes into play. The word “polymerase” is used to describe an enzyme that binds individual small units (nucleotides, in the case of DNA) together to form a long, repeating chain or polymer (a DNA strand). As we have learned, a molecule of DNA is a polymer made of multiple individual units called nucleotides.

Key Term: DNA Polymerase

DNA polymerase is an enzyme that adds nucleotides complementary to the template strand to synthesize a new strand. This enzyme plays an essential role in DNA replication.

DNA polymerase generates a new strand of DNA along each original strand by adding nucleotides to the new strand, ensuring that the rules of complementary base-pairing, which we learned about earlier, are followed. To build the new strand of DNA, the DNA polymerase uses a pool of free-floating nucleotides that remain available in the cell. Figure 5 represents a simple diagram of the action of DNA polymerase.

The DNA polymerase enzyme is a highly efficient one it synthesizes complementary strands very rapidly and with a high level of accuracy. Another important feature of this enzyme is that it can only synthesize a new strand of DNA in the

direction. This feature poses a problem in a dividing cell. You might be wondering how! Well, let’s take a look at a replication fork in a strand of DNA. As we know, the two strands of DNA run antiparallel to one another. As you can see in Figure 6, the strand at the top of the image has a

open end at the fork, while the strand at the bottom has a

As new strands of DNA are synthesized, they must also run antiparallel to their complementary original strand. Although the two new strands are synthesized simultaneously, let’s first consider the formation of a new strand along the strand at the top of the figure, bearing in mind the fact that DNA polymerase can only synthesize a new strand in the

direction. In this case, a new strand is able to form continuously, without any interruptions or breaks.

Let’s now consider the strand at the bottom. As the DNA molecule unwinds, DNA polymerase encounters the

end of this strand. This would require a new complementary strand to be synthesized in the

direction, which DNA polymerase cannot do!

The DNA polymerase works around this problem by moving further along the strand, as shown in the figure, and synthesizing a short fragment of new DNA in the

direction, toward the open end of the replication fork. It then moves further along, behind this fragment, and does the same, synthesizing another short fragment. As the enzyme progresses along the strand, a discontinuous complementary strand begins to take shape. In Figure 8, you can see how this happens.

These fragments are called Okazaki fragments. If you now take a look at the molecule of DNA, you can see that along one template strand, a new strand is formed continuously, in the same direction as the opening fork, with no breaks. This template strand is therefore called the “leading strand.” The opposite template strand is called the “lagging strand,” since the new strand of DNA is synthesized discontinuously in fragments.

Key Term: Leading Strand

In DNA replication, the leading strand is the strand of DNA along which the new strand is synthesized continuously, in the same direction as the fork.

Key Term: Lagging Strand

In DNA replication, the lagging strand is the strand of DNA along which the new strand is synthesized discontinuously, in fragments.

Key Term: Okazaki Fragments

Okazaki fragments are the short fragments of DNA that are synthesized discontinuously along the lagging strand during DNA replication.

Another enzyme, DNA ligase, is responsible for joining these fragments together along the lagging strand. As you can see in Figure 9, DNA ligase moves along the fragmented strand, joining or “ligating” the fragments together by forming new phosphodiester bonds between one fragment and the next.

Key Term: DNA Ligase

DNA ligase is an enzyme that can join the gaps between the sugar–phosphate backbone of DNA by forming a phosphodiester bond.

Example 2: Understanding the Role of DNA Ligase

In semiconservative DNA replication, what is the primary role of DNA ligase?

  1. DNA ligase adds nucleotides to a growing DNA chain to synthesize a strand of DNA complementary to the template strand.
  2. DNA ligase joins the backbones of fragments formed on a complementary strand during replication.
  3. DNA ligase catalyzes the breaking of phosphodiester bonds in the sugar–phosphate backbone, so the DNA can be split into fragments that are ready for replication.
  4. DNA ligase breaks the hydrogen bonds between base pairs, separating the two strands of DNA that are ready for replication.
  5. DNA ligase joins RNA primers to the

Answer

When a molecule of DNA replicates, it unwinds so as to separate the two individual strands. Along each of these strands, a new strand of DNA is synthesized by the enzyme DNA polymerase, following the rules of complementary base-pairing.

We know that each strand of DNA has a

end and that the two strands of DNA in a DNA molecule must always run antiparallel, or in opposite directions, to one another. When synthesizing a new strand of DNA, DNA polymerase is only capable of adding nucleotides in the

Because of this feature of DNA polymerase, the two DNA strands in the molecule are replicated using two different methods. Along the “leading strand,” whose

end is at the opening of the replication fork, DNA polymerase can synthesize a continuous, unbroken complementary DNA strand. Along the “lagging strand,” whose

end is at the opening of the replication fork, DNA polymerase must instead synthesize short fragments of DNA in the

direction, as shown in the figure.

The fragments formed along the lagging strand are called Okazaki fragments. In order for the fragments to be functional, they must be joined together to form one long, continuous strand of newly synthesized DNA. This function is accomplished by DNA ligase that joins the sugar–phosphate backbones of adjacent fragments through phosphodiester bonds.

Let’s now take a look at the options provided in the question. The sentence that best fits the information we now have about DNA ligase is “DNA ligase joins the backbones of fragments formed on a complementary strand during replication.” This is therefore the right answer.

We have now been over the roles of the three important enzymes involved in DNA replication and have understood the mechanism of this process. Figure 10 shows a simple overview of how the whole process takes place and what the result of this process will be.

Let’s take a close look at each of the new DNA molecules on the right side of Figure 10. You may notice that each new DNA molecule contains one original strand and one newly synthesized strand. Because of this feature, the process of DNA replication is called “semiconservative replication”: the older strands of DNA are conserved as the molecule of DNA replicates.

Key Term: Semiconservative Replication

Semiconservative replication describes the mechanism of DNA replication in all living cells, in which each new DNA molecule is composed of one original strand of DNA and one newly synthesized strand of DNA.

Earlier on in this explainer, we talked about the genetic code formed by the sequence of nitrogenous bases along a strand of mRNA, which is synthesized from DNA. When a cell “reads” the sequence, it can interpret this information to produce specific proteins, which then go on to control the characteristics of the organism. Sections of DNA that contain a sequence of bases that code for a specific protein are called genes, which is a word you might have heard before.

Although the process of DNA replication is accurate and highly efficient, it is not completely error free. What would happen if, during DNA replication, the DNA polymerase were to make an error in adding a new nucleotide to the new strand? At one point, instead of adding a complementary nucleotide according to rules of base-pairing, what if a different nitrogenous base were accidentally added? Can you think about what this would mean?

On the newly synthesized DNA strand, this specific point in the strand would change the genetic code. You can think of this as something similar to a spelling mistake in a sentence. These errors, which are called mutations, can sometimes have serious consequences. Let’s consider a quick example. You might remember learning about hemoglobin, the molecule that carries oxygen in our blood. If the gene that codes for hemoglobin is mutated, the hemoglobin protein will be produced incorrectly. This can cause a condition called sickle cell anemia, which distorts the shape of the red blood cells in the body.

Key Term: Mutation

A mutation is an error or an alteration in a sequence of nucleotides.

In order to prevent such errors from arising during DNA replication, the enzyme DNA polymerase performs another crucial function. As it adds nucleotides to the growing new strand, it also “proofreads” or checks its own work. In this way, if the wrong nucleotide has accidentally been added, DNA polymerase will identify the error and swap the wrong nucleotide for the right one! It does this through exonuclease activity, which means it removes incorrect nucleotides and replaces them with correct ones. You can see an example of this in Figure 11.

As we learned earlier, DNA ligase is an enzyme that joins fragments of DNA together by forming new phosphodiester bonds, linking the fragments. When DNA is physically damaged, causing breaks in the strands, DNA ligase can function as a DNA repair enzyme. It uses the complementary strand of the DNA double helix as a template to form new phosphodiester bonds.

DNA repair, therefore, depends on the presence of two strands carrying the genetic information. When one strand is damaged, the intact information on the complementary strand can be used by DNA repair enzymes to replace the damaged sections. This is why it is so important for DNA replication to be an accurate, error-free process!

Example 3: Understanding how Proofreading Eliminates Errors During DNA Replication

When errors in DNA replication occur, the newly formed strand can be proofread and be recognized as not being complementary to the original strand. Which enzyme is responsible for correcting these errors during replication?

Answer

When a cell undergoes division, its DNA replicates itself, so as to provide each new cell with a copy of DNA which can control the cell’s characteristics and functions. We know that a single molecule of DNA is composed of two complementary polynucleotide strands. Each of these strands is made up of multiple individual units called nucleotides, which form a sequence of nitrogenous bases. The order or sequence of nitrogenous bases along a strand of DNA encodes the “genetic information” that needs to be replicated when a cell divides.

The enzyme DNA polymerase synthesizes new strands of DNA that are complementary to each of the original strands, by following the rules of complementary base-pairing: adenine binds to thymine through two hydrogen bonds, and guanine binds to cytosine through three hydrogen bonds.

The genetic information carried in these strands is crucially important to the normal functioning of a cell, as it forms a genetic code that provides the cell with instructions for the production of proteins. The process of DNA replication, though accurate, is not foolproof, which means that sometimes errors can arise in the new sequence, when a noncomplementary nitrogenous base is accidentally added.

Every word in the English language has a specific meaning. If a word is written down with a spelling mistake in it, the meaning of this word would be lost! This is similar to what happens when errors arise in a DNA sequence, which are also called mutations. Mutations in DNA can lead to several different diseases and disorders.

In order to prevent mutations during DNA replication, the enzyme DNA polymerase “proofreads” its own work, checking that each new nucleotide is complementary to the original strand as it goes along. If it detects an error, or a noncomplementary nucleotide, it quickly replaces this with the correct one!

The answer to this question is, therefore, DNA polymerase.

Let’s summarize the key points we have learned from this explainer.

Key Points

  • When a living cell divides, its DNA must replicate so that each new cell receives a copy of DNA.
  • DNA replication is a semiconservative process.
  • In order for a molecule of DNA to replicate, the two strands must first unwind. DNA helicase is responsible for this and “unzips” the DNA molecule by breaking the hydrogen bonds between the nitrogenous bases.
  • DNA polymerase synthesizes new strands of DNA along each template strand by adding complementary nucleotides to the chain.
  • DNA polymerase can only synthesize DNA in the