5. DNA Replication, Repair, and Recombination

deamination of cytosine

Makes Uracil.

topoisomerase I

Making single-stranded DNA breaks to relieve supercoiling at origin type I DNA topoisomerase with tyrosine at the active site→ the two ends of the DNA double helix can now rotate relative to each other, relieving accumulated strain

crossing over

Process in which homologous chromosomes exchange portions of their chromatids during meiosis.

telomeres

Repeated DNA sequences at the ends of eukaryotic chromosomes.

Okazaki fragments

Small fragments of DNA produced on the lagging strand during DNA replication, joined later by DNA ligase to form a complete strand.

DNA-only transposons

Type of transposable element that exists as DNA throughout its life cycle. Many types move by cut-and-paste transposition

Nucleotide Excision Repair (NER)

a DNA repair system in which several nucleotides in the damaged strand are removed from the DNA and the undamaged strand is used as a template to resynthesize a normal strand. In this path- way, a large multienzyme complex scans the DNA for a distortion in the double helix, rather than for a specific base change. Once it finds a lesion, it cleaves the phosphodiester backbone of the abnormal strand on both sides of the distortion, and a DNA helicase peels away the single-strand oligonucleotide containing the lesion. The large gap produced in the DNA helix is then repaired by DNA polymerase and DNA ligase

Homologous recombination in meiosis starts with a bold stroke:

a specialized protein (called Spo11 in budding yeast) breaks both strands of the DNA double helix in one of the recombining chromosomes

Single-strand DNA-binding (SSB) proteins,

also called helix-destabilizing proteins, bind tightly and cooperatively to exposed single-strand DNA without covering the bases, which therefore remain available as templates. These proteins are unable to open a long DNA helix directly, but they aid helicases by stabilizing the unwound, single-strand conformation.

The fraction of damaged genes underestimates the actual mutation rate because

any mutations are silent (for exam- ple, those that change a codon but not the amino acid it specifies, or those that change an amino acid without affecting the activity of the protein coded for by the gene).

DNA Damage Can Be Removed by More Than One Pathway

base excision repair Nucleotide Excision Repair (NER)

strand-directed mismatch repair.

corrects errors made during replication

DNA-only transposons can relocate from a donor site to a target site by

cut- and-paste transposition been found to operate in developing immune systems. vertebrates, catalyzing the DNA rearrangements that produce antibody and T cell receptor diversity. Known as V(D)J recombination,

Mobile Genetic Elements (MGEs)

discrete segments of DNA that move as units from one location to another within other DNA molecules

Double-Strand Breaks Are Efficiently Repaired by :

nonhomologous end joining, homologous recombination,

DNA primase,

synthesizes a short RNA primer to provide a 3'-OH group for the attachment of DNA nucleotides

exonucleolytic proofreading,

takes place immediately after those rare instances in which an incorrect nucle- otide 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

However, as attractive as this model might be, the DNA polymerases at replication forks can synthesize only in

the 5ʹ-to-3ʹ direction.

Homologous Recombination (HR)

the DNA is repaired using the sister chromatid as a template. ccurately corrects these accidents and, because they occur during nearly every round of DNA replication, this repair mechanism is essential for every proliferating cel

Depurination

the loss of a purine base from a nucleotide

mutation rate

the probability that a gene will mutate when a cell divides

phase variation

the routine switching on and off of certain genes The DNA inversion changes the orientation of a promoter (a DNA sequence that directs transcription of a gene) that is located within the inverted DNA segment

human cell loses about 18,000 purine bases (adenine and guanine) every day because

their N-glycosyl linkages to deoxyribose hydrolyze, a spontaneous reaction called depurination. Similarly, a spontaneous deamination of cytosine to uracil in DNA occurs at a rate of about 100 bases per cell per day

branch migration

whereby DNA is spooled through the Holliday junction by continually breaking and re-forming base pairs

DNA replication requires the cooperation of many proteins. These include:

(1) DNA polymerase and DNA primase to catalyze nucleoside triphosphate polymer- ization; (2) DNA helicases and single-strand DNA-binding (SSB) proteins to help in opening up the DNA helix so that it can be copied; (3) DNA ligase and an enzyme that degrades RNA primers to seal together the discontinuously synthesized lagging- strand DNA fragments; and (4) DNA topoisomerases to help to relieve helical winding and DNA tangling problems. Many of these proteins associate with each other at a replication fork to form a highly efficient "replication machine, " through which the activities and spatial movements of the individual components are coordinated.

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.

Once a topoisomerase II molecule binds to such a crossing site, the protein uses ATP hydrolysis to perform the following set of reactions efficiently:

(1) it breaks one double helix revers- ibly to create a DNA "gate"; (2) it causes the second, nearby double helix to pass through this opening; and (3) it then reseals the break and dissociates from the DNA.

Figure 5-18 A bacterial replication fork.

(A) This schematic diagram shows 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 (Movie 5.4 and Movie 5.5). (B) An electron micrograph showing the replication machine from the bacteriophage T4 as it moves along a template synthesizing DNA behind it. (C) An interpretation of the micrograph is given in the sketch: note especially the DNA loop on the lagging strand. Apparently, the replication proteins became partly detached from the very front of the replication fork during the preparation of this sample for electron microscopy. (B, courtesy of Jack Griffith; see P .D. Chastain et al., J. Biol. Chem. 278:21276-21285, 2003.)

transposons

(jumping genes) short strands of DNA capable of moving from one location to another within a cell's genetic material

histone chaperones

- assist the assembly of histone octamers on the DNA - bind the H3 - H4 tetramer or H2A-H2B dimer

replication fork

A Y-shaped region on a replicating DNA molecule where new strands are growing.

mutation

A change in a gene or chromosome.

lagging strand.

A discontinuously synthesized DNA strand that elongates by means of Okazaki fragments, each synthesized in a 5' to 3' direction away from the replication fork.

DNA ligase

A linking enzyme essential for DNA replication; catalyzes the covalent bonding of the 3' end of a new DNA fragment to the 5' end of a growing chain.

DNA synthesis catalyzed by DNA polymerase.

A) DNA polymerase catalyzes the stepwise addition of a deoxyribonucleotide to the 3ʹ-OH end of a polynucleotide chain, the growing primer strand that is paired to an existing template strand. The newly synthesized DNA strand therefore polymerizes in the 5ʹ-to-3ʹ direction as shown also in the previous figure. Because 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. (B) Structure of DNA polymerase complexed wth DNA (orange), as determined by x-ray crystallography (Movie 5.1). The template DNA strand is the longer strand and the newly synthesized DNA is the shorter. (C) Schematic diagram of DNA polymerase, based on the structure in (B). 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 (middle diagram (C)). 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.

Switching gene expression by DNA inversion in bacteria.

Alternating transcription of two flagellin genes in a Salmonella bacterium is caused by a conservative site-specific recombination event that inverts a small DNA segment containing a promoter. (A) In one orientation, the promoter activates transcription of the H2 flagellin gene as well as that of a repressor protein that blocks the expression of the H1 flagellin gene. Promoters and repressors are described in detail in Chapter 7; here we note simply that a promoter is needed to express a gene into protein and that a repressor blocks this from happening. (B) When the promoter is inverted, it no longer turns on H2 or the repressor, and the H1 gene, which is thereby released from repression, is expressed instead. The inversion reaction requires specific DNA sequences (red) and a recombinase enzyme that is encoded in the invertible DNA segment. This site-specific recombination mechanism is activated only rarely (about once in every 105 cell divisions). Therefore, the production of one or the other flagellin tends to be faithfully inherited in each clone of cells.

topoisomerase II,

An enzyme that breaks a DNA double helix, rotates the ends, and seals the break.

Telomerase

An enzyme that catalyzes the lengthening of telomeres in eukaryotic germ cells. _______ 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 (Figure 5-33). 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

DNA helicases

An enzyme that unwinds the DNA double helix during DNA replication

Shelterin complex

Binds to the end of the telomere Involved in formation of heterochromatin and T-loop (TRF2) Contains 6 proteins: -POT1 -TPP1 -TIN2 -TRF1 -TRF2 -Rap1

DNA repair

Collective term for the enzymatic processes that correct deleterious changes affecting the continuity or sequence of a DNA molecule.

Base Excision Repair (BER)

Corrects DNA containing a damaged DNA base, involves a battery of enzymes called DNA glycosylases, each of which can recognize a specific type of altered base in DNA and catalyze its hydrolytic removal. The "missing tooth" created by DNA glycosylase action is recognized by an enzyme called AP endonuclease (AP for apurinic or apyrimidinic, endo to signify that the nuclease cleaves within the polynucleotide chain), which cuts the phosphodiester backbone, after which the resulting gap is repaired

DNA topoisomerases.

Create a nick in the helix to relieve supercoils created during replication. *Fluoroquinolones inhibit DNA gyrase (a specific prokaryotic topoisomerase)

transposase

Cuts DNA backbone, leaving single-stranded "sticky ends"

Rad51

DNA binding ATPase involved in DNA recombination repair

Editing by DNA polymerase.

DNA polymerase complexed with the DNA template in the polymerizing mode (left) and the editing mode (right). The catalytic sites for the exonucleolytic (E) and the polymerization (P) reactions are indicated. In the editing mode, the newly synthesized DNA transiently unpairs from the template and enters the editing site where the most recently added nucleotide is catalytically removed.

cut and paste transposition

DNA-only transposons can be recognized in chromosomes by the "inverted repeat DNA sequences" (red) present at their ends. These sequences, which can be as short as 20 nucleotides, are all that is necessary for the DNA between them to be transposed by the particular transposase enzyme associated with the element. The cut-and-paste movement of a DNA-only transposable element from one chromosomal site to another begins when the transposase brings the two inverted DNA sequences together, forming a DNA loop. Insertion into the target chromosome, also catalyzed by the transposase, occurs at a random site through the creation of staggered breaks in the target chromosome (purple arrowheads). Following the transposition reaction, the single-strand gaps created by the staggered breaks are repaired by DNA polymerase and ligase (black). As a result, the insertion site is marked by a short direct repeat of the target DNA sequence, as shown. Although the break in the donor chromosome (green) is repaired, this process often alters the DNA sequence, causing a mutation at the original site of the excised transposable element (not shown).

On the basis of their structure and transposition mechanism, transposons can be grouped into three large classes:

DNA-only transposons, retroviral-like retrotransposons, and nonretroviral retrotransposons.

Alleles

Different forms of a gene

, DNA polymerase,

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

The DNA double helix seems optimal for repair. As noted above, it contains a backup copy of all genetic information. Equally importantly, the nature of the four bases in DNA makes the distinction between undamaged and damaged bases very clear.

For example, every possible deamination event in DNA yields an "unnatural" base, which can be directly recognized and removed by a specific DNA glycosylase. Hypoxanthine, for example, is the simplest purine base capable of pairing specifically with C, but hypoxanthine is the direct deamination prod- uct of A

sliding clamp

Holds DNA polymerase in place during strand extension

The need for accuracy probably explains why DNA replication occurs only in the 5ʹ-to-3ʹ direction.

If there were a DNA polymerase that added deoxyribonucleo- side triphosphates in the 3ʹ-to-5ʹ direction, the growing 5ʹ end of the chain, rather than the incoming mononucleotide, would have to provide the activating triphos- phate needed for the covalent linkage. n this case, the mistakes in polymeriza- tion could not be simply hydrolyzed away, because the bare 5ʹ end of the chain thus created would immediately terminate DNA synthesis

Enzyme-catalyzed branch movement at a Holliday junction by branch migration.

In E. coli, a tetramer of the RuvA protein (green) and two hexamers of the RuvB protein (yellow) bind to the open form of the junction. The RuvB protein, which resembles the hexameric helicases used in DNA replication (Figure 5-14), uses the energy of ATP hydrolysis to spool DNA rapidly through the Holliday junction, extending the heteroduplex region as shown. The RuvA protein coordinates this movement, threading the DNA strands to avoid tangling. (PDB codes: 1IXR, 1C7Y.)

Loss of heterozygosity (LOH)

In a cell, the loss of normal function in one allele of a gene where the other allele is already inactivated by mutation.

Mechanism of double-strand break repair by homologous recombination.

In essence, the broken DNA duplex and the template duplex carry out a "strand dance" so that one of the damaged strands can use the complementary strand of the intact DNA duplex as a template for repair. First, the ends of the broken DNA are chewed back, or "resected, " by specialized nucleases to produce overhanging, single-strand 3ʹ ends. The next step is strand exchange (also called strand invasion), during which one of the single-strand 3ʹ ends from the damaged DNA molecule worms its way into the template duplex and searches it for homologous sequences through base-pairing. We describe this remarkable reaction in detail in the next section. Once stable base-pairing is established (which completes the strand exchange step), an accurate DNA polymerase extends the invading strand by using the information provided by the undamaged template molecule, thus restoring the damaged DNA. The last steps—strand displacement, further repair synthesis, and liga- tion—restore the two original DNA double helices and complete the repair process.

Structure of a portion of telomerase.

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. (Modified from J. Lingner and T.R. Cech, Curr. Opin. Genet. Dev. 8:226-232, 1998. With permission from Elsevier.)

When a nucleosome is traversed by a replication fork, the histone octamer appears to be broken into an H3-H4 tetramer and two H2A-H2B dimers

The H3-H4 tetramer remains loosely associated with DNA and is distributed at random to one or the other daughter duplex, but the H2A-H2B dimers are released completely from DNA. Freshly made H3-H4 tetramers are added to the newly synthesized DNA to fill in the "spaces, " and H2A-H2B dimers—half of which are old and half new—are then added at random to complete the nucleosomes Histones are synthesized mainly in S phase, when the level of histone mRNA increases about fiftyfold as a result of both increased transcription and decreased mRNA degradation.

Why might an erasable RNA primer be preferred to a DNA primer that would not need to be erased?

The argument that a self-correcting polymerase cannot start chains de novo also implies the converse: an enzyme that starts chains anew cannot be efficient at self-correction. Thus, any enzyme that primes the synthesis of Okazaki fragments will of necessity make a relatively inaccurate copy (at least one error in 105). Even if the copies retained in the final product constituted as little as 5% of the total genome (for example, 10 nucleotides per 200-nucleotide DNA fragment), the resulting increase in the overall mutation rate would be enor- mous.

"double-check" the exact base-pair geometry before it catalyzes

The correct nucleotide has a higher affinity for the moving polymerase than does the incorrect nucleotide, because the correct pairing is more energetically favorable. Moreover, after nucleotide binding, but before the nucleotide is covalently added to the growing chain, the enzyme must undergo a conformational change in which its "grip" tightens around the active site (see Figure 5-4). Because this change occurs more readily with correct than incorrect base-pairing, it allows the polymerase to "double-check" the exact base-pair geometry before it catalyzes the addition of the nucleotide. Incorrectly paired nucleotides are harder to add and therefore more likely to diffuse away before the polymerase can mistakenly add them.

Holliday junction or cross-strand exchange

The initially formed structure (A) is usually drawn with two strands crossing, as in Figure 5-54. An isomerization of the Holliday junction (B) produces an open, symmetrical structure that is bound by specialized proteins. (C) These proteins "move" the Holliday junctions by a coordinated set of branch- migration reactions (see Figure 5-57 and Movie 5.8). (D) Structure of the Holliday junction in the open form depicted in (B). The Holliday junction is named for the scientist who first proposed its formation. (PDB code: 1DCW.)

The proteins that initiate DNA replication in bacteria.

The mechanism shown was established by studies in vitro with mixtures of highly purified proteins. For E. coli DNA replication, the major initiator 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 helicase- loading 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).

leading strand

The new continuous complementary DNA strand synthesized along the template strand in the mandatory 5' to 3' direction.

Two types of DNA rearrangement produced by conservative site-specific recombination.

The only difference between the reactions in (A) and (B) is the relative orientation of the two short DNA sites (indicated by arrows) at which a site-specific recombination event occurs. (A) Through an integration reaction, a circular DNA molecule can become incorporated into a second DNA molecule; by the reverse reaction (excision), it can exit to re-form the original DNA circle. Many bacterial viruses move in and out of their host chromosomes in this way. (B) Conservative site-specific recombination can also invert a specific segment of DNA in a chromosome. A well-studied example of DNA inversion through site-specific recombination occurs in the bacterium Salmonella typhimurium, an organism that is a major cause of food poisoning in humans; as described in the following section, the inversion of a DNA segment changes the type of flagellum that is produced by the bacterium.

Therefore, it is not surprising that the initiation of DNA replication is highly regulated.

The process begins when initiator proteins (in their ATP-bound state) bind in multiple copies to specific DNA sites located at the replication origin, wrapping the DNA around the proteins to form a large protein-DNA complex that destabilizes the adjacent double helix. This complex then attracts two DNA helicases, each bound to a helicase loader, and these are placed around adjacent DNA single strands whose bases have been exposed by the assembly of the initiator protein-DNA complex. The helicase loader is analogous to the clamp loader we encountered above; it has the additional job of keeping the helicase in an inactive form until it is properly loaded onto a nascent replication fork. Once the helicases are loaded, the loaders dissociate and the helicases begin to unwind DNA, exposing enough single-strand DNA for DNA primase to synthesize the first RNA primers

clamp loader

The three-dimensional structure of the clamp protein, determined by x-ray diffraction, revealed it to be a large ring around the DNA double helix. One face of the ring binds to the back of the DNA polymerase, and the whole ring slides freely along the DNA as the polymerase moves. The assembly of the clamp around the DNA requires ATP hydrolysis by a special protein complex,

strand-directed mismatch repair system

The two proteins shown are present in both bacteria and eukaryotic cells: MutS binds specifically to a mismatched base pair, while MutL scans the nearby DNA for a nick. Once MutL finds a nick, it triggers the degradation of the nicked strand all the way back through the mismatch. Because nicks are largely confined to newly replicated strands in eukaryotes, replication errors are selectively removed. In bacteria, an additional protein in the complex (MutH) nicks unmethylated (and therefore newly replicated) GATC sequences, thereby beginning the process illustrated here. In eukaryotes, MutL contains a DNA nicking activity that aids in the removal of the damaged strand.

All of a cell's DNA is under constant surveillance for damage, and the repair mechanisms we have described act on all parts of the genome. However, cells have a way of directing DNA repair to the DNA sequences that are most urgently needed.

They do this by linking RNA polymerase, the enzyme that transcribes DNA into RNA as the first step in gene expression, to the nucleotide excision repair pathway. As discussed above, this repair system can correct many different types of DNA damage. RNA polymerase stalls at DNA lesions and, through the use of coupling proteins, directs the excision repair machinery to these sites.

nonretroviral retrotransposons

Transposition of the L1 element (red) begins when an endonuclease attached to the L1 reverse transcriptase (green) and the L1 RNA (blue) nick the target DNA at the point at which insertion will occur. This cleavage releases a 3ʹ-OH DNA end in the target DNA, which is then used as a primer for the reverse transcription step shown. This generates a single-strand DNA copy of the element that is directly linked to the target DNA. In subsequent reactions, further processing of the single-strand DNA copy results in the generation of a new double-strand DNA copy of the L1 element that is inserted at the site of the initial nick.

By contrast, the RNA polymerase enzymes involved in gene transcription do not need such an efficient exonucleolytic proofreading mechanism:

errors in making RNA are not passed on to the next generation, and the occasional defective RNA molecule that is produced has no long-term significance. RNA polymerases are thus able to start new polynucleotide chains without a primer.

prereplicative complex.

formed by ORC, Cdc6, and helicase; DNA is ready to replicate on S phase is signaled

Nonhomologous end joining (NHEJ)

in which the broken ends are simply brought together and rejoined by DNA ligation, generally with the loss of nucleo- tides at the site of joining because there seems to be no mechanism to ensure that two ends being joined were originally next to each other in the genome, nonhomologous end joining can occasionally generate rearrangements in which one broken chromo- some becomes covalently attached to another. This can result in chromosomes with two centromeres and chromosomes lacking centromeres altogether; both types of aberrant chromosomes are missegregated during cell division.

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.

transpositional recombination

is a process in which a mobile element is inserted into a target DNA.

conservative site-specific recombination (CSSR)

is a type of genetic recombination in which DNA strand exchange takes place between segments possessing at least a certain degree of sequence homology. Enzymes known as site-specific recombinases (SSRs) perform rearrangements of DNA segments by recognizing and binding to short, specific DNA sequences (sites), at which they cleave the DNA backbone, exchange the two DNA helices involved, and rejoin the DNA strands.

DNA renaturation or hybridization,

occurs when a rare random collision juxtaposes complementary nucleotide sequences on two matching DNA single strands, allowing the formation of a short stretch of double helix between them. This relatively slow helix-nucleation step is followed by a very rapid "zippering" step, as the region of double helix is extended to maximize the number of base-pairing interactions

gene conversion

process of nonreciprocal genetic exchange that can produce abnormal ratios of gametes following meiosis enetic studies show that only small sections of DNA typically undergo gene conversion, and in many cases only a part of a gene is changed.

Retroviral-like retrotransposons

repeated sequences that have the same directionality but at different ends of the transposon. The first step in their trans- position is the transcription of the entire transposon, producing an RNA copy of the element that is typically several thousand nucleotides long. This transcript, which is translated as a messenger RNA by the host cell, encodes a reverse tran- scriptase enzyme. This enzyme makes a double-strand DNA copy of the RNA molecule via an RNA-DNA hybrid intermediate, precisely mirroring the early stages of infection by a retrovirus

translesion polymerases

replicate across the damage and generate a rough draft of the damaged sequence Human cells have seven translesion polymerases, some of which can recog- nize a specific type of DNA damage and correctly add the nucleotide required to restore the initial sequence. Despite their usefulness in allowing heavily damaged DNA to be replicated, these translesion polymerases do, as noted above, pose risks to the cell. They are probably responsible for most of the base-substitution and single-nucleotide deletion mutations that accumulate in genomes; although they generally produce mutations when copying damaged DNA

Heteroduplex region

segment of DNA molecule located between the two breakpoints mark sites of potential gene conversion—where the four haploid chromosomes produced by meiosis contain three copies of a DNA sequence from one homolog and only one copy of this sequence from the other homolog

The importance of DNA repair is evident from the large investment that cells make in the enzymes that carry it out:

several percent of the coding capacity of most genomes is devoted solely to DNA repair functions.

WHAT WE DON'T KNOW DNA Replication, Repair, and Recombination

• How does DNA replication contend with all the other processes that occur simultaneously on chromosomes, including DNA repair and gene transcription? • What is the basis for the low frequency of errors in DNA replication observed in all cells? Is this the best that cells can do given the speed of replication and the limits of molecular diffusion? Was this mutation rate selected in evolution to provide genetic variation? • Cells have only one fundamental way of replicating DNA but many different ways of repairing it. Are there still other, undiscovered ways that cells have for repairing DNA? • Do the many "dead" transposons in the human genome provide any benefits to humans?