Chapter 5: DNA Replication, Repair, and Recombination

. Most DNA sequences that can serve as an origin of replication are found to contain

(1) a binding site for a large, multisubunit initiator protein called ORC, for origin recognition complex; (2) a stretch of DNA that is rich in As and Ts and therefore easy to melt; and (3) at least one binding site for proteins that facilitate ORC binding, probably by adjusting chromatin structure.

Exonucleolytic proofreading by DNA polymerase during DNA replication

1. C transiently base-pairs with A and is incorporated by DNA polymerase into the primer strand 2. unpaired 3′-OH end of primer blocks further elongation of primer strand by DNA polymerase 3. 3′-to-5′ exonuclease activity attached to DNA polymerase chews back to create a basepaired 3′-OH end on the primer strand 4. DNA polymerase resumes the process of adding nucleotides to the base-paired 3′-OH term-21end of the primer strand

Sliding Clamp Mechanism

1. The loader binds to a free clamp molecule, forcing a gap in its ring of subunits so that this ring is able to slip around DNA. 2. The clamp loader, thanks to its screw-nut structure, recognizes the region of DNA that is double-stranded and latches onto it, tightening around the complex of a template strand with a freshly synthesized elongating (primer) strand. 3. It carries the clamp along this double-stranded region until it encounters the 3ʹ end of the primer, at which point the loader hydrolyzes ATP and releases the clamp, allowing it to close around the DNA and bind to DNA polymerase. In the simplified reaction shown here, the clamp loader dissociates into solution once the clamp has been assembled. At a true replication fork, the clamp loader remains close to the polymerase so that, on the lagging strand, it is ready to assemble a new clamp at the start of each new Okazaki fragment

The Synthesis of many DNA fragments on the lagging strand

1. new RNA primer synthesis by DNA primase 2. DNA polymerase adds to new RNA primer to start new Okazaki fragment 3.DNA polymerase finishes DNA fragment 4. old RNA primer erased and replaced by DNA 5. sealing by DNA ligase joins new Okazaki fragment to the growing chain

Replication fork

A Y-shaped region on a replicating DNA molecule where new strands are growing. The replication fork contains a multienzyme complex that contains DNA polymerase which synthesizes the DNA of both new daughter strands

A bacterial replication Fork

A current view of the arrangement of replication proteins at a replication fork when DNA is being synthesized. The lagging-strand DNA is folded to bring the lagging-strand DNA polymerase molecule into a complex with the leading-strand DNA polymerase molecule. This folding also brings the 3ʹ end of each completed Okazaki fragment close to the start site for the next Okazaki fragment. Because the lagging-strand DNA polymerase molecule remains bound to the rest of the replication proteins, it can be reused to synthesize successive Okazaki fragments. In this diagram, it is about to let go of its completed DNA fragment and move to the RNA primer that is just being synthesized. Additional proteins (not shown) help to hold the different protein components of the fork together, enabling them to function as a well-coordinated protein machine

Eukaryotic Chromosomes Contain Multiple Origins of Replication

Because of the much greater size of most eukaryotic chromosomes, a different strategy is required to allow their replication in a timely manner. Because of the much greater size of most eukaryotic chromosomes, a different strategy is required to allow their replication in a timely manner. genes are being expressed. Approximately 30,000- 50,000 origins of replication are used each time a human cell divides. (2) The human genome has many more (perhaps tenfold more) potential origins than this, and different cell types use different sets of origins. This may allow a cell to coordinate its active origins with other features of its chromosomes such as which The excess origins also provide "backups" in case a primary origin fails. (3) As in bacteria, replication forks are formed in pairs and create a replication bubble as they move in opposite directions away from a common point of origin, stopping only when they collide head-on with a replication fork moving in the opposite direction or when they reach a chromosome end. In this way, many replication forks operate independently on each chromosome and yet form two complete daughter DNA helices

Replication Fork Structure

Both daughter DNA strands are synthesized in the 5'-3' prime direction. Because of this the DNA synthesized on the lagging strand must be made initially as a series of fragments. The fragments are synthesized sequentially with those nearest to the strand being synthesized first

New Nucleosomes Are Assembled Behind the Replication Fork

Chromosome replication requires not only the duplication of DNA but also the synthesis and assembly of new chromosomal proteins onto the DNA behind each replication fork. Parental H3-H4 tetramers are distributed at random to the daughter DNA molecules, with roughly equal numbers inherited by each daughter. In contrast, H2A-H2B dimers are released from the DNA as the replication fork passes. This release begins just in front of the replication fork and is facilitated by chromatin remodeling complexes that move with the fork. Histone chaperones (NAP1 and CAF1) restore the full complement of histones to daughter molecules using both parental and newly synthesized histones. Although some daughter nucleosomes contain only parental histones or only newly synthesized histones, most are hybrids of old and new

DNA helicases

DNA helicase binds to a single strand of DNA at the origin of replication and hydrolyzes ATP. It then uses that energy to propel itself along the strand. When it encounters a double strand it continues to move along the strand and rips the helix apart therby prying it apart at 1000 nucleotide pairs per second.

Editing by DNA Polymerase

DNA polymerase functions as a "self-correcting" enzyme that removes its own polymerization errors as it moves along the DNA

DNA synthesis process

DNA synthesis catalyzes the addition of a deoxyribose nucleotide to 3'OH end of a polynucleotide chain, the growing primer strand that is paired to an existing template. The newly synthesized DNA strand therefore polymerizes in the 5'-3' direction. Each incoming deoxyribonucleoside triphosphate must pair with the template strand to be recognized by the DNA polymerase, this strand determines which of the four possible deoxyribonucleotides (A, C, G, or T) will be added. The reaction is driven by a large, favorable free-energy change, caused by the release of pyrophosphate and its subsequent hydrolysis to two molecules of inorganic phosphate. The template DNA strand is the longer strand and the newly synthesized DNA is the shorter. The proper base-pair geometry of a correct incoming deoxyribonucleoside triphosphate causes the polymerase to tighten around the base pair, thereby initiating the nucleotide addition reaction. Dissociation of pyrophosphate relaxes the polymerase, allowing translocation of the DNA by one nucleotide so the active site of the polymerase is ready to receive the next deoxyribonucleoside triphosphate.

DNA topoisomerase I mechanism

DNA topoisomerase covalently attaches to a DNA phosphate, thereby breaking a phosphodiester linkage in one DNA strand 2. the two ends of the DNA double helix can now rotate relative to each other, relieving accumulated strain 3. the original phosphodiester bond energy is stored in the phosphotyrosine linkage, making the reaction reversible 4. spontaneous re-formation of the phosphodiester bond regenerates both the DNA helix and the DNA topoisomerase

DNA polymerase

Enzyme involved in DNA replication that joins individual nucleotides to produce a DNA molecule

DNA Winding Problem

For a bacterial replication fork moving at 500 nucleotides per second, the parental DNA helix ahead of the fork must rotate at 50 revolutions per second. (B) If the ends of the DNA double helix remain fixed (or difficult to rotate), tension builds up in front of the replication fork as it becomes overwound. Some of this tension can be taken up by supercoiling, whereby the DNA double helix twists around itself \. However, if the tension continues to build up, the replication fork will eventually stop because further unwinding requires more energy than the helicase can provide.

What would happen if the proofreading system simply recognized a mismatch in newly replicated DNA and randomly corrected one of the two mismatched nucleotides?

If the proofreading system simply recognized a mismatch in newly replicated DNA and randomly corrected one of the two mismatched nucleotides, it would mistakenly "correct" the original template strand to match the error exactly half the time, thereby failing to lower the overall error rate. To be effective, such a proofreading system must be able to distinguish and remove the mismatched nucleotide only on the newly synthesized strand, where the replication error occurred.

In Eukaryotes, DNA Replication Takes Place During Only One Part of the Cell Cycle

In a mammalian cell, the S phase typically lasts for about 8 hours; in simpler eukaryotic cells such as yeasts, the S phase can be as short as 40 minutes. By its end, each chromosome has been replicated to produce two complete copies, which remain joined together at their centromeres until the M phase (M for mitosis), which soon follows

DNA replication is semiconservative

In a round of replication, each of the two strands of DNA is used as a template for the formation of a complementary DNA strand. The original strands therefore remain intact through many cell generations

Different Regions on the Same Chromosome Replicate at Distinct Times in S Phase

In mammalian cells, the replication of DNA in the region between one replication origin and the next should normally require only about an hour to complete, given the rate at which a replication fork moves and the largest distances measured between replication origins. Yet S phase usually lasts for about 8 hours in a mammalian cell. This implies that the replication origins are not all activated simultaneously; indeed, replication origins are activated in clusters of about 50 adjacent replication origins, each of which is replicated during only a small part of the total S-phase interval. It seems that the order in which replication origins are activated depends, in part, on the chromatin structure in which the origins reside. We saw in Chapter 4 that heterochromatin is a particularly condensed state of chromatin, while euchromatin, where most transcription occurs, has a less condensed conformation. Heterochromatin tends to be replicated very late in S phase, suggesting that the timing of replication is related to the packing of the DNA in chromatin. Once initiated, however, replication forks seem to move at comparable rates throughout S phase, so the extent of chromosome condensation seems to influence the time at which replication forks are initiated, rather than their speed once formed.

mutator genes

Increase the rate of spontaneous mutation

sliding clamp

Keeps the polymerase firmly on the DNA when moving, but releases it as soon as the polymerase runs into a double-strand region of DNA.

Strand-directed mismatch repair in Eukaryotes

Newly synthesized lagging-strand DNA transiently contains nicks (before they are sealed by DNA ligase) and such nicks (also called single-strand breaks) provide the signal that directs the mismatch proofreading system to the appropriate strand. This strategy also requires that the newly synthesized DNA on the leading strand be transiently nicked. . Fortunately, most of us inherit two good copies of each gene that encodes a mismatch proofreading protein; this protects us, because it is highly unlikely for both copies to become mutated in the same cell.

Single-strand DNA-binding proteins (SSBPs)

Once you get the DNA apart they might have short regions that can base pair with themselves. Once the dna is separated by helicase single strand binding proteins can hold DNA in place and stop the strand from binding to itself. Bind tightly and cooperatively to exposed single-stranded DNA without covering the bases. They aid helicases by stabilizing the unwound single strand conformation. They also coat and straighten out the regions of the single strand which occurs routinely in the lagging strand. This prevents the formation of the short hairpin helices that regularly form in single strand DNA

DNA topoisomerase

Relives additional coiling ahead of replication fork. A DNA topoisomerase can be viewed as a reversible nuclease that adds itself covalently to a DNA backbone phosphate, thereby breaking a phosphodiester bond in a DNA strand. This reaction is reversible, and the phosphodiester bond re-forms as the protein leaves.

telomerase

Replenishes telomeres each time a cell divides. Telomerase recognizes the tip of an existing telomere DNA repeat sequence and elongates it in the 5ʹ-to-3ʹ direction, using an RNA template that is a component of the enzyme itself to synthesize new copies of the repeat

The structure of DNA helicase

Six identical subunits bind and hydrolyze ATP in an ordered fashion to propel this molecule, like a rotary engine, along a DNA single strand that passes through the central hole

Telomerase structure

Telomerase is a large protein- RNA complex. The RNA (blue) contains a templating sequence for synthesizing new DNA telomere repeats. The synthesis reaction itself is carried out by the reverse transcriptase domain of the protein, shown in green. A reverse transcriptase is a special form of polymerase enzyme that uses an RNA template to make a DNA strand; telomerase is unique in carrying its own RNA template with it. Telomerase also has several additional protein domains (not shown) that are needed to assemble the enzyme at the ends of chromosomes. The enzymatic portion of telomerase resembles other reverse transcriptases, proteins that synthesize DNA using an RNA template, although, in this case, the telomerase RNA also contributes functional groups to make the catalysis more efficient. After extension of the parental DNA strand by telomerase, replication of the lagging strand at the chromosome end can be completed by the conventional DNA polymerases, using these extensions as a template to synthesize the complementary strand

telomerase replication

The 3ʹ end of the parental DNA strand is extended by RNA-templated DNA synthesis; this allows the incomplete daughter DNA strand that is paired with it to be extended in its 5ʹ direction. This incomplete, lagging strand is presumed to be completed by DNA polymerase α, which carries a DNA primase as one of its subunits

leading strand

The DNA daughter strand that is synthesized continuously

Telomere length regulation

The cells corrects the telomerase to its desired length over time

The importance of Strand-Directed mismatch repair

The importance of mismatch proofreading in humans is seen in individuals who inherit one defective copy of a mismatch repair gene (along with a functional gene on the other copy of the chromosome). These people have a marked predisposition for certain types of cancers.

DNA Replication in Eukaryotes vs Bacteria

There are more protein components in eukaryotic replication machines than there are in the bacterial analogs, even though the basic functions are the same. Thus, for example, the eukaryotic single-strand binding (SSB) protein is formed from three subunits, whereas only a single subunit is found in bacteria. Similarly, the eukaryotic DNA primase is incorporated into a multisubunit enzyme that also contains a polymerase called DNA polymerase α-primase. This protein complex begins each Okazaki fragment on the lagging strand with RNA and then extends the RNA primer with a short length of DNA. At this point, the two main eukaryotic replicative DNA polymerases, Polδ and Polε, come into play: Polδ completes each Okazaki fragment on the lagging strand and Polε extends the leading strand. The increased complexity of eukaryotic replication machinery probably reflects DNA REPLICATION MECHANISMS. The DNA-helix-passing reaction catalyzed by DNA topoisomerase II. Unlike type I topoisomerases, type II enzymes hydrolyze ATP , which is needed to release and reset the enzyme after each cycle. Type II topoisomerases are largely confined to proliferating cells in eukaryotes; partly for that reason, they have been effective targets for anticancer drugs. Some of these drugs inhibit topoisomerase II at the third step in the figure and thereby produce high levels of double-strand breaks that kill rapidly dividing cells. The phosphates in the DNA backbone that become covalently bonded to the topoisomerase. two circular DNA double helices that are interlocked two circular DNA double helices that are separated topoisomerase recognizes the entanglement and makes a reversible covalent attachment to the two opposite strands of one of the double helices creating a doublestrand break and forming a protein gate the topoisomerase gate opens to let the second DNA helix pass the gate shuts releasing the red helix reversal of the covalent attachment of the topoisomerase restores an intact double helix Chapter 5: DNA Replication, Repair, and Recombination more elaborate controls. For example, the orderly maintenance of different cell types and tissues in animals and plants requires that DNA replication be tightly regulated. Moreover, eukaryotic DNA replication must be coordinated with the elaborate process of mitosis

How telomeres helps

There is always a little bit of single stranded DNA. Which means your DNA is broken. The t loop wraps around the end of a chromosome making it appear to be double strandedThere is always a little bit of single stranded DNA. Which means your DNA is broken. The t loop wraps around the end of a chromosome making it appear to be double stranded

DNA ligase

This enzyme seals a broken phosphodiester bond. As DNA ligase uses a molecule of ATP to activate the 5ʹ end at the nick (step 1) before forming the new bond (step 2). In this way, the energetically unfavorable nick-sealing reaction is driven by being coupled to the energetically favorable process of ATP hydrolysis

Strand-directed mismatch repair in E.coli

This system depends on the methylation of selected A residues in DNA. Methyl groups are added to all A residues in the sequence GATC, but not until some time after the A has been incorporated into a newly synthesized DNA chain. As a result, the only GATC sequences that have not yet been methylated are in the new strands just behind a replication fork. The recognition of these unmethylated GATCs allows the new DNA strands to be transiently distinguished from old ones, as required if their mismatches are to be selectively removed. The three-step process involves recognition of a newly synthesized strand, excision of the portion containing the mismatch, and resynthesis of the excised segment using the old strand as a template

DNA Syntheis Begins at Replication Origins

To begin DNA replication, the double helix must first be opened up and the two strands separated to expose unpaired bases. As we shall see, the process of DNA replication is begun by special initiator proteins that bind to double-strand DNA and pry the two strands apart, breaking the hydrogen bonds between the bases.The positions at which the DNA helix is first opened are called replication origins. In simple cells like those of bacteria or yeast, origins are specified by DNA sequences several hundred nucleotide pairs in length. This DNA contains both short sequences that attract initiator proteins and stretches of DNA that are especially easy to open. We saw in Figure 4-4 that an A-T base pair is held together by fewer hydrogen bonds than a G-C base pair. Therefore, DNA rich in A-T base pairs is relatively easy to pull apart, and regions of DNA enriched in A-T base pairs are typically found at replication origins.

Strand-directed mismatch repair

detects the potential for distortion in the DNA helix from the misfit between noncomplementary base pairs.

, how is the process regulated to ensure that all the DNA is copied once and only once

during G1 phase, the replicative helicases are loaded onto DNA next to ORC to create a prereplicative complex. Then, upon passage from G1 phase to S phase, specialized protein kinases come into play to activate the helicases. The resulting opening of the double helix allows the loading of the remaining replication proteins, including the DNA polymerases. The protein kinases that trigger DNA replication simultaneously prevent assembly of new prereplicative complexes until the next M phase resets the entire cycle. They do this, in part, by phosphorylating ORC, rendering it unable to accept new helicases. This strategy provides a single window of opportunity for prereplicative complexes to form (G1 phase, when kinase activity is low) and a second window for them to be activated and subsequently disassembled (S phase, when kinase activity is high). Because these two phases of the cell cycle are mutually exclusive and occur in a prescribed order, each origin of replication can fire once and only once during each cell cycle.

Sliding Ring

holds a moving DNA polymerase onto the DNA

clamp loader

hydrolyzes ATP as it loads the clamp on to a primer-template junction

An example of Strand-Directed mismatch repaired gone wrong

in a type of colon cancer called hereditary nonpolyposis colon cancer (HNPCC), spontaneous mutation of the one functional gene produces a clone of somatic cells that, because they are deficient in mismatch proofreading, accumulate mutations unusually rapidly. Most cancers arise in cells that have accumulated multiple mutations and cells deficient in mismatch proofreading therefore have a greatly enhanced chance of becoming cancerous.

Proteins that initiate DNA replication in bacteria

itiator protein, the helicase, and the primase are the dnaA, dnaB, and dnaG proteins, respectively. In the first step, several molecules of the initiator protein bind to specific DNA sequences at the replication origin and destabilize the double helix by forming a compact structure in which the DNA is tightly wrapped around the protein. Next, two helicases are brought in by helicaseloading proteins (the dnaC proteins), which inhibit the helicases until they are properly loaded at the replication origin. Helicase-loading proteins prevent the replicative DNA helices from inappropriately entering other single-strand stretches of DNA in the bacterial genome. Aided by single-strand binding protein (not shown), the loaded helicases open up the DNA, thereby enabling primases to enter and synthesize initial primers. In subsequent steps, two complete replication forks are assembled at the origin and move off in opposite directions. The initiator proteins are displaced as the left-hand fork moves through them (not shown)

Topoisomerase I

produces a transient single-strand break; this break in the phosphodiester backbone allows the two sections of DNA helix on either side of the nick to rotate freely relative to each other, using the phosphodiester bond in the strand opposite the nick as a swivel point. Any tension in the DNA helix will drive this rotation in the direction that relieves the tension. As a result, DNA replication can occur with the rotation of only a short length of helix—the part just ahead of the fork. Because the covalent linkage that joins the DNA topoisomerase protein to a DNA phosphate retains the energy of the cleaved phosphodiester bond, resealing is rapid and does not require additional energy input. In this respect, the rejoining mechanism differs from that catalyzed by the enzyme DNA ligase, discussed previously

RNA primers

synthesizes a short polynucleotide in the 5ʹ-to-3ʹ direction and then stops, making the 3ʹ end of this primer available for the DNA polymerase.Because an RNA primer contains a properly base-paired nucleotide with a 3ʹ-OH group at one end, it can be elongated by the DNA polymerase at this end to begin an Okazaki fragment. The synthesis of each Okazaki fragment ends when this DNA polymerase runs into the RNA primer attached to the 5ʹ end of the previous fragment

exonuclease activity

takes place immediately after those rare instances in which an incorrect nucleotide is covalently added to the growing chain. DNA polymerase enzymes are highly discriminating in the types of DNA chains they will elongate: they require a previously formed, base-paired 3ʹ-OH end of a primer strand. This 3ʹ-to-5ʹ proofreading exonuclease clips off any unpaired or mispaired residues at the primer terminus, continuing until enough nucleotides have been removed to regenerate a correctly base-paired 3ʹ-OH terminus that can prime DNA synthesis. In this way, DNA polymerase functions as a "self-correcting" enzyme that removes its own polymerization errors as it moves along the DN

lagging strand

the daughter strand that is synthesized discontinuously.the direction of nucleotide polymerization is opposite to the overall direction of DNA chain growth

Bacterial Chromosomes Typically Have a Single Origin of DNA Replication

the interaction of the initiator protein with the replication origin is carefully regulated, with initiation occurring only when sufficient nutrients are available for the bacterium to complete an entire round of replication. Initiation is also controlled to ensure that only one round of DNA replication occurs for each cell division. After replication is initiated, the initiator protein is inactivated by hydrolysis of its bound ATP molecule, and the origin of replication experiences a "refractory period." The refractory period is caused by a delay in the methylation of newly incorporated A nucleotides in the origin. Initiation cannot occur again until the A's are methylated and the initiator protein is restored to its ATP-bound state.

DNA templating

the mechanism the cell uses to copy the nucleotide sequence of one DNA strand into a complementary strand. This process requires the seperation of the DNA helix into two template strands, and entails the recognition of each nucleotide in the DNA template strands by a free (unpolymerized) complementary nucleotide. The DNA helix exposes the hydrogen-bond donor and acceptor groups on each DNA base for base-pairing with the appropriate incoming free nucleotide, aligning it for its enzyme-catalyzed polymerization into a new DNA chain.

DNA primase

uses ribonucleoside triphosphates to synthesize short RNA primers on the lagging strand