Biochemistry Chapter 34

DNA synthesis has the following characteristics

1. The reaction requires all four activated precursors—that is, the deoxynucleoside 5′-triphosphates dATP, dGTP, dCTP, and TTP—as well as the Mg2+ ion. 2. The new DNA strand is assembled directly on a preexisting DNA template. DNA polymerases catalyze the formation of a phosphodiester linkage efficiently only if the base on the incoming nucleoside triphosphate is complementary to the base on the template strand. Thus, DNA polymerase is a template-directed enzyme that synthesizes a product with a base sequence complementary to that of the template. 3. DNA polymerases require a primer to begin synthesis. A primer strand having a free 3′-OH group must be already bound to the template strand. The strand-elongation reaction catalyzed by DNA polymerases is a nucleophilic attack by the 3′-OH end of the growing strand on the innermost phosphorus atom of deoxynucleoside triphosphate (Figure 34.2). A phosphodiester bridge is formed and pyrophosphate is released. The subsequent hydrolysis of pyrophosphate to yield two ions of orthophosphate (Pi) by pyrophosphatase helps drive the polymerization forward. Elongation of the DNA strand proceeds in the 5′-to-3′ direction. 4. Many DNA polymerases are able to correct mistakes in DNA by removing mismatched nucleotides. These polymerases have a distinct nuclease activity that allows them to excise incorrect bases by a separate reaction. For instance, DNA polymerase I has three distinct active sites: the polymerase site, a 3′ → 5′ exonuclease site, and a 5′ → 3′ exonuclease site. The 3′ → 5′ nuclease activity contributes to the remarkably high fidelity of DNA replication, which has an error rate of less than 10−8 per base pair. We will consider the function of the 5′ → 3′ nuclease activity shortly.

Both the Okazaki fragments and the leading strand are synthesized in the

5′ → 3′ direction. The discontinuous assembly of the lagging strand enables 5′ → 3′ polymerization at the nucleotide level to give rise to overall growth in the 3′ → 5′ direction.

How is this primer formed?

An important clue came from the observation that RNA synthesis is required for the initiation of DNA synthesis. In fact, RNA primes the synthesis of DNA. A specialized RNA polymerase called primase joins the prepriming complex in a multisubunit assembly called the primosome. Primase synthesizes a short stretch of RNA (about ten nucleotides) that is complementary to one of the template DNA strands (Figure 34.13). The RNA primer is removed by a 5′ → 3′ exonuclease; in E. coli, the exonuclease is present as the third active site in DNA polymerase I.

Two distinct polymerases are needed to copy a eukaryotic replicon...which are..

An initiator polymerase called polymerase α begins replication. This enzyme includes a primase subunit, used to synthesize the RNA primer, as well as an active DNA polymerase. After this polymerase has added a stretch of about 20 deoxynucleotides to the primer, it is replaced by DNA polymerase α, a more-processive enzyme and the principal replicative polymerase in eukaryotes. This process is called polymerase switching because one polymerase has replaced another.

Most naturally occurring DNA molecules are negatively supercoiled. What is the basis for this prevalence?

As discussed in Chapter 33, negative supercoiling arises from the unwinding or underwinding of DNA. In essence, negative supercoiling prepares DNA for processes requiring separation of the DNA strands, such as replication. The presence of supercoils in the immediate area of unwinding would, however, make unwinding difficult (Figure 34.8). Therefore, negative supercoils must be continuously removed, and the DNA relaxed, as the double helix unwinds.

The Specificity of Replication Is Dictated by the

Complementarity of Bases

As replication proceeds, these fragments ( Okazaki fragments) become covalently joined through the action of

DNA ligase

The full replication machinery in a cell comprises more than 20 proteins engaged in intricate and coordinated interplay. The key enzymes are called

DNA polymerase

The gaps between fragments of the nascent lagging strand are filled by

DNA polymerase I. This essential enzyme also uses its 5′ → 3′ exonuclease activity to remove the RNA primer lying ahead of the polymerase site. The primer cannot be erased by DNA polymerase III, because the enzyme lacks 5′ → 3′ editing capability.

Even with the DNA template exposed and the prepriming complex assembled, there are still obstacles to DNA synthesis.

DNA polymerases can add nucleotides only to a free hydroxyl group; they cannot start a strand de novo. Therefore, a primer is required.

The mode of synthesis of the lagging strand is necessarily more complex. As mentioned earlier, the lagging strand is synthesized in fragments so that 5′ → 3′ polymerization leads to overall growth in the 3′ → 5′ direction. Yet the synthesis of the lagging strand is coordinated with the synthesis of the leading strand. How is this coordination accomplished?

Examination of the subunit composition of the DNA polymerase III holoenzyme reveals an elegant solution (Figure 34.18). The holoenzyme includes two copies of the polymerase core enzyme, which consists of the DNA polymerase itself, a 3′-to-5′ proofreading exonuclease (see p. 599), two copies of the dimeric β2-subunit sliding clamp, and several other proteins. The core enzymes are linked to a central structure, which consists of the clamp loader, and subunits that interact with the single-strand-binding protein. The entire central structure interacts with helicase DnaB.

How does the enzyme sense whether a newly added base is correct?

First, an incorrect base will not pair correctly with the template strand and will be unlikely to be linked to the new strand. Second, even if an incorrect base is inserted into the new strand, it is likely to be deleted. After the addition of a new nucleotide, the DNA is pulled by one base pair into the enzyme. If an incorrect base is incorporated, the enzyme stalls owing to the structural disruption caused by the presence of a non-Watson-Crick base pair in the enzyme, and the pause provides additional time for the strand to wander into the exonuclease site. There is a cost to this editing function, however: DNA polymerase I removes approximately 1 correct nucleotide in 20. Although the removal of correct nucleotides is slightly wasteful energetically, proofreading increases the accuracy of replication by a factor of approximately 1000.

Whereas the genomes of essentially all bacteria are circular, the chromosomes of human beings and other eukaryotes are linear. The free ends of linear DNA molecules introduce several complications.

First, the unprotected termini of the DNA at the end of a chromosome are likely to be more susceptible to digestion by exonucleases if they are left to freely dangle at the end of the chromosome during replication. Second, the complete replication of DNA ends is difficult because polymerases act only in the 5′ → 3′ direction. The lagging strand would have an incomplete 5′ end after the removal of the RNA primer. Each round of replication would further shorten the chromosome, which would, like the Cheshire cat, slowly disappear

proofreading

Many polymerases further enhance the fidelity of replication by the use of proofreading mechanisms. One polymerase from E. coli, DNA polymerase I used in DNA replication and repair, displays an exonuclease activity in addition to the polymerase activity. The exonuclease removes mismatched nucleotides from the 3′ end of DNA by hydrolysis. If the wrong nucleotide is inserted, the malformed product is not held as tightly in the polymerase active site. It is likely to flop about because of the weaker hydrogen bonding and to find itself in the exonuclease active site, where the trespassing nucleotide is removed (Figure 34.10). This flopping is the result of Brownian motion

Assembly of DnaA and formation of the prepriming complex

Monomers of DnaA bind to their binding sites (shown in yellow) in oriC and come together to form a complex structure, possibly the cyclic hexamer shown here. This structure marks the origin of replication and favors DNA strand separation in the AT-rich sites (green). (B) The AT-rich regions are unwound by DnaB and trapped by the single-stranded-binding protein (SSB). At this stage, the complex is ready for the synthesis of the RNA primers and assembly of the DNA polymerase III holoenzyme.

The two strands of DNA comparison

One Strand of DNA Is Made Continuously and the Other Strand Is Synthesized in Fragments. Recall that the two strands are antiparallel; that is, they run in opposite directions. On cursory examination, both daughter strands appear to grow in the same direction, as shown in Figure 34.14. However, this appearance of same-direction growth presents a conundrum, because all known DNA polymerases synthesize DNA in the 5′ → 3′ direction but not in the 3′ → 5′ direction. How then does one of the daughter DNA strands appear to grow in the 3′ → 5

Replication in eukaryotes is mechanistically similar to replication in bacteria but is more challenging in three ways.

One challenge is sheer size: E. coli must replicate 4.6 million base pairs, whereas a human diploid cell must replicate 6 billion base pairs. The second challenge is the fact that the genetic information for E. coli is contained on 1 chromosome, whereas, in human beings, 23 pairs of chromosomes must be replicated. Finally, whereas the E. coli chromosome is circular, human chromosomes are linear. Because of the nature of DNA synthesis on the lagging strand, linear chromosomes are subject to shortening with each round of replication unless countermeasures are taken.

How does DNA become trapped inside the sliding clamp?

Polymerases also include assemblies of subunits that function as clamp loaders. These enzymes grasp the sliding clamp and, utilizing the energy of ATP, pull apart the two subunits of the sliding clamp on one side. DNA can move through the gap, inserting itself through the central hole. ATP hydrolysis then releases the clamp, which closes around the DNA.

How are replicons controlled so that each replicon is replicated only once in each cell division?

Proteins, called licensing factors because they permit (license) the formation of the DNA synthesis initiation complex, bind to the origin of replication. These proteins ensure that each replicon is replicated once and only once in each round of DNA synthesis. After the licensing factors have established the initiation complex, these factors are subsequently destroyed. The license expires after one use.

The Separation of DNA Strands Requires

Specific Helicases and ATP Hydrolysis

So if telomerase activity is low in human cells, why can cancer, which is characterized by unlimited cell proliferation, develop in humans?

Telomerase reactivation is one of the hallmarks of cancer cells. Because cancer cells express high levels of telomerase, whereas most normal cells do not, telomerase is a potential target for anticancer therapy. A variety of approaches for blocking telomerase expression or blocking its activity are under investigation for cancer treatment and prevention.

Telomeres Are Replicated by

Telomerase, a Specialized Polymerase That Carries Its Own RNA Template

What is the basis of DNA polymerase's low error rate?

The answer to this question is complex, but one important factor is induced fit—the change in the structure of the enzyme when it binds the correct nucleotide. DNA polymerases close down around the incoming nucleoside triphosphate (dNTP), as shown in Figure 34.5. The binding of a dNTP into the active site of a DNA polymerase triggers a conformational change: the finger domain rotates to form a tight pocket into which only a properly shaped base pair will readily fit.

DNA polymerase structure

The first DNA polymerase structure determined was that of a fragment of E. coli DNA polymerase I called the Klenow fragment. Notice that, like other DNA polymerases, the polymerase unit resembles a right hand with fingers , palm , and thumb. The Klenow fragment also includes an exonuclease domain that removes incorrect nucleotide bases.

holoenzyme DNA polymerase III

The hallmarks of this multisubunit assembly are not only its fidelity, but also its very high catalytic potency and processivity. The holoenzyme catalyzes the formation of many thousands of phosphodiester linkages before releasing its template, compared with only 20 for DNA polymerase I. The DNA polymerase III holoenzyme grasps its template and does not let go until the template has been completely replicated. Another distinctive feature of the holoenzyme is its catalytic prowess: 1000 nucleotides are added per second compared with only 10 per second for DNA polymerase I. This acceleration is accomplished with no loss of accuracy. The greater catalytic prowess of polymerase III is largely due to its processivity; no time is lost in repeatedly stepping on and off the template.

Typical structures of the helicases

The helicases in DNA replication are typically oligomers containing six subunits that form a ring structure. Each subunit has a loop that extends toward the center of the ring structure and interacts with DNA. A possible mechanism for the action of a helicase is shown in Figure 34.7. Two subunits are bound to ATP, two to ADP and Pi, and two are intially free of nucleotides. One of the strands of the double helix passes through the hole in the center of the helicase, bound to the loops on two adjacent subunits, one of which has bound ATP and the other of which has bound ADP + Pi.

What is the trombone model

The lagging-strand template is looped out so that it passes through the polymerase site in one subunit of polymerase III in the 3′ → 5′ direction. After adding about 1000 nucleotides, DNA polymerase III lets go of the lagging-strand template by releasing the sliding clamp. A new loop is then formed, a sliding clamp is added, and primase again synthesizes a short stretch of RNA primer to initiate the formation of another Okazaki fragment. This mode of replication has been termed the trombone model because the size of the loop lengthens and shortens like the slide on a trombone . The replication of the leading and lagging strands is coordinated by the looping out of the lagging strand to form a structure that acts somewhat as a trombone slide does, growing as the replication fork moves forward. When the polymerase on the lagging strand reaches a region that has been replicated, the sliding clamp is released and a new loop is formed.

What characteristics of the chromosome ends, called telomeres (from the Greek telos, meaning "an end"), mitigate these two problems? ( problems meaning the free ends of linear DNA)

The most-notable feature of telomeric DNA is that it contains hundreds of tandem repeats of a hexanucleotide sequence. One of the strands is G-rich at the 3′ end, and it is slightly longer than the other strand. In human beings, the repeating G-rich sequence is AGGGTT. This simple repeat precludes the likelihood that the sequence encodes any information, but the simple repeat does facilitate the formation of large duplex loops (Figure 34.22). The single-stranded region at the very end of the structure has been proposed to loop back to form a DNA duplex with another part of the repeated sequence, displacing a part of the original telomeric duplex. This looplike structure is formed and stabilized by specific telomere-binding proteins. Such structures would nicely protect the end of the chromosome from degradation.

We now see how telomeres help to protect the ends of the DNA, but the problem of how the ends are replicated remains. What steps are taken to prevent the lagging strand from disappearing after repeated cycles of replication?

The solution is to add nucleotides to the leading strand so that the lagging strand will always maintain its approximate length. This task falls to the enzyme telomerase, which contains an RNA molecule that acts as a template for extending the leading strand. The extended leading strand acts as a template to elongate the lagging strand, thus ensuring that the chromosome will not shorten after many rounds of replication

How then does one of the daughter DNA strands appear to grow in the 3′ → 5′ direction?

This dilemma was resolved when careful experimentation found that a significant proportion of newly synthesized DNA exists as small fragments. These units of about a thousand nucleotides, called Okazaki fragments (after Reiji Okazaki, who first identified them), are present briefly in the vicinity of the replication fork (Figure 34.15).

In E. coli, DNA replication starts at a unique site within the entire 4 × 106 bp genome.

This origin of replication, called the oriC locus, is a 245-bp region that has several unusual features

Functions of type 1 and 2 of topoisomerase

Type I topoisomerases catalyze the relaxation of supercoiled DNA, a thermodynamically favorable process. Type II topoisomerases utilize free energy from ATP hydrolysis to add negative supercoils to DNA. In bacteria, type II topoisomerase is called DNA gyrase.

DNA ligase

an enzyme that uses ATP hydrolysis to power the joining of DNA fragments to form one of the daughter strands.

Novobiocin

blocks the binding of ATP to gyrase.

Werner syndrome

causes by a disfunction of the helicase, and leads people to premature aging

DNA polymerases catalyze the step-by-step addition of

deoxyribonucleotide units to a DNA strand

Studies show that a nucleotide with a base that is very similar in shape to adenine but lacks the ability to form base-pairing hydrogen bonds can still

direct the incorporation of thymidine, both in vitro and in vivo (Figure 34.4). However, DNA polymerases replicate DNA even more faithfully than can be accounted for by these interactions alone.

Nalidixic acid and ciprofloxacin

interfere with the breakage and rejoining of DNA strands. These two gyrase inhibitors are widely used to treat urinary-tract and other infections.

DNA gyrase

is the target of several antibiotics that inhibit this bacterial topoisomerase much more than the eukaryotic one

The strand formed from Okazaki fragments is termed the WHAT where the other strand is called the WHAT

lagging strand, whereas the one synthesized without interruption is the leading strand.

Telomeres Are Unique Structures at the Ends of

linear chromosomes

Ciprofloxacin

more commonly known as "cipro," became a "celebrity" in the United States, owing to the anthrax poisonings in the fall of 2001 (Figure 34.9). It is a potent broad-spectrum antibiotic that prevents anthrax poisoning by preventing the growth of Bacillus anthracis if taken early enough after infection.

The first two challenges ( size and number of chromosomes) are met by the use of

multiple origins of replication, which are located between 30 and 300 kilobase pairs (kbp) apart. In human beings, replication of the entire genome requires about 30,000 origins of replication, with each chromosome containing several hundred. Each origin of replication represents a replication unit, or replicon. The use of multiple origins of replication requires mechanisms for ensuring that each sequence is replicated once and only once.

DNA must be locally unwound to expose single-stranded templates for replication. This unwinding puts a strain on the molecule by causing the

overwinding of nearby regions.

DNA ligase catalyzes the formation of a

phosphodiester linkage between the 3′-hydroxyl group at the end of one DNA chain and the 5′-phosphate group at the end of the other (Figure 34.20). ATP is commonly used as the energy source to drive this thermodynamically uphill reaction.

The binding of DnaA molecules begins the building of the

replication complex. Additional proteins, such as DnaB, then join DnaA. The DnaB protein is a helicase that utilizes ATP hydrolysis to unwind the duplex, including the AT-rich regions. Single-strand-binding protein (SSB) then binds to the newly generated single-stranded regions, preventing the two complementary strands from re-forming the double helix. The result of this process is the generation of a structure called the prepriming complex, which makes single-stranded DNA accessible for other enzymes to begin the synthesis of the complementary strands.

The site of DNA synthesis is called the

replication fork because the complex formed by the newly synthesized daughter strands arising from the parental duplex resembles a two-pronged fork

Enzymes called topoisomerases introduce or eliminate

supercoils by temporarily cleaving DNA.

Structure of holoenzyme DNA polymerase III

the dimeric β2 subunit of DNA polymerase III forms a ring that surrounds the DNA duplex. Notice the central cavity through which the DNA template slides. Clasping the DNA molecule in the ring, the polymerase enzyme is able to move without falling off the DNA substrate.

Specific enzymes, termed helicases, utilize

the energy of ATP hydrolysis to power strand separation.

The binding of the dNTP containing the proper base is favored by the formation of a base pair, which is stabilized by specific hydrogen bonds. The binding of a noncomplementary base is less likely because

the interactions are energetically weaker.

The term processivity refers

to the ability of an enzyme to catalyze many consecutive reactions without releasing its substrate.

Topoisomerases Prepare the Double Helix for

unwinding

DNA polymerases

which promote the formation of the bonds joining units of the DNA backbone.

The source of the processivity —the ability to stay "on task" for the holoenzyme DNA polymerase III—is the

β2 subunit, which has the form of a star-shaped ring (Figure 34.16). A 35-Å-diameter hole in its center can readily accommodate a duplex DNA molecule yet leaves enough space between the DNA and the protein to allow rapid sliding and turning in the course of replication. A catalytic rate of 1000 nucleotides polymerized per second requires the sliding of 100 turns of duplex DNA (a length of 3400 Å, or 0.34 μm) through the central hole of β2 per second. Thus, β2 plays a key role in replication by serving as a sliding DNA clamp.