LEHNINGER PRINCIPLES OF BIOCHEMISTRY - VOL. 3. INFORMATION PATHWAYS - 2017

PART III. INFORMATION PATHWAYS

25. DNA METABOLISM

25.2. DNA Repair

Most Cells contain only a single or double set of genomic DNA. Damaged Proteins and RNA molecules can be rapidly replaced using the information encoded in DNA, but DNA molecules themselves cannot be replaced. Maintaining the integrity of information in DNA is a critical cellular task, carried out by a complex array of DNA repair systems. DNA can be damaged by various processes; some are spontaneous, while others are catalyzed by environmental factors (see Chapter 8, Vol. 1). Even the Replication process itself can occasionally distort the information contained in DNA if errors lead to incorrect base pairing (e.g., a G-T pair).

The chemistry of DNA damage is diverse and complex. The cellular response to specific types of damage involves a wide range of enzyme systems that catalyze some of the most fascinating chemical transformations in DNA METABOLISM. We will first examine the consequences of Changes in DNA sequence and then explore specific repair systems.

Cancers Are Linked to Mutations

The best way to illustrate Structure/19.html">The Importance of DNA repair is to consider the consequences of unrepaired damage. The most dangerous DNA changes are those that become permanent As a result of replication and transmission to new generations of cells. A stable change in The nucleotide sequence of DNA is called a mutation. Mutations may involve the replacement of one base pair with another (single-nucleotide substitutions) or the insertion and deletion of one or more Base Pairs (insertions and deletions). If a mutation affects a nonessential region of DNA or is of no consequence to Gene function, it is termed a silent mutation. Occasionally, mutations provide their host with a biological advantage. In most cases, however, manifested mutations are either neutral or deleterious.

In mammals, There is a clear correlation between the accumulation of mutations and Cancer. A simple test developed by Bruce Ames can detect the ability of a chemical compound to induce specific, easily detectable mutations in special bacterial strains (Fig. 25-21). According to this test, very few of the substances we use daily are mutagens. However, over 90% of compounds that exert carcinogenic effects on animals under experimental conditions show mutagenic activity in the Ames test. Given this strong correlation between mutagenesis and carcinogenesis, the Ames test for bacterial mutagens is widely used as a rapid and inexpensive method for identifying human carcinogens.

Class="center">Fig. 25-21 The Ames test for identifying carcinogens based on mutagenicity. A strain of Salmonella typhimurium carrying a mutation that inactivates an enzyme in the Histidine biosynthetic pathway is plated on a medium lacking histidine. Only isolated cells grow. (a) A few small S. typhimurium colonies that manage to grow on histidine-free medium carry spontaneous back-mutations that restore histidine Biosynthesis. Three plates (b, c, and d) containing this medium are inoculated with an equal number of cells. Filter paper disks soaked in various concentrations of the mutagen are then placed in the center of each plate. The mutagen significantly increases the probability of reverse mutation and, consequently, the number of grown colonies. A clear zone around the filter paper indicates that the mutagen concentration there is high enough to be lethal to cells. As the mutagen diffuses away from the filter paper disk, its concentration drops to sublethal levels that induce reverse mutations. Mutagens can be compared based on their effect on mutation frequency. Because many compounds undergo various chemical transformations upon entering The Cell, compounds are sometimes tested for mutagenicity after preliminary incubation with a Liver extract. Some substances display mutagenicity only after such Treatment.

Thousands of lesions accumulate in The Genome of a typical mammalian cell over a 24-hour period. However, thanks to DNA repair, fewer than one lesion in 1,000 becomes a mutation. The DNA molecule is relatively stable, but in the absence of repair systems, the cumulative effect of many infrequent but destructive reactions could lead to organismal death. ■

All Cells Have Multiple DNA Repair Systems

The number and variety of repair systems reflect the importance of DNA repair for cell survival, as well as the diverse ways in which DNA can be damaged (Table 25-5). Some common types of damage, such as pyrimidine dimers (see Fig. 8-31), can be repaired by multiple pathways. Unlike most metabolic pathways, where every ATP molecule is tightly budgeted and used optimally, many DNA repair processes are energetically very inefficient. But when the integrity of Genetic information is at stake, energetic costs do not matter.

DNA repair is largely possible because the DNA molecule consists of two complementary strands. Damage to DNA in one strand can be removed and accurately corrected by using the undamaged complementary strand as a template. Below, we examine The major types of repair systems, starting with the repair of rare mismatched NUCLEOTIDES left over after replication.

Mismatch Repair.

The correction of rare mismatched bases remaining after replication in E. coli increases overall replication fidelity by another 2 to 3 orders of magnitude. Mismatched bases are almost always corrected According to the information in the old (template) strand, so the repair system must somehow distinguish between the original and newly synthesized strands. To accomplish this, the cell labels the template DNA with methyl groups. The E. coli mismatch repair system involves at least 12 protein components (Table 25-5) that either discriminate between strands or participate directly in the repair process.

Table 25-5. Types of DNA Repair Systems in E. coli

The Mechanism of strand discrimination has not been established for most Bacteria and eukaryotes, but it is well understood in E. coli and related bacteria. In these prokaryotes, strand discrimination relies on the action of Dam methylase, which, as you recall, methylates DNA at the N⁶ position of adenine residues within (5’) GATC sequences. Immediately following the passage of the Replication fork, there is a brief period (seconds or minutes) during which the template strand is methylated while the newly synthesized strand is not (Fig. 25-22). Transiently unmethylated GATC sequences in the newly synthesized strand allow it to be distinguished from the template strand. Misincorporated bases near a hemimethylated GATC sequence are removed based on the information contained in the methylated parental (template) strand. In vitro studies have shown that if GATC sequences are methylated on both strands, very few errors are corrected; if neither strand is methylated, repair still occurs, but neither strand has the advantage. Cell methylation-based repair systems efficiently eliminate single-nucleotide substitutions up to 1,000 bp away from a hemimethylated GATC sequence.

Fig. 25-22. Methylation and mismatch repair. Methylation of DNA strands in E. coli can serve to distinguish parental (template) strands from newly synthesized strands, which is critical for correcting single-nucleotide substitutions; see Fig. 25-23. Methylation occurs at the N⁶ position of adenine residues in 5’-(GATC) sequences. This sequence is a palindrome (see Fig. 8-18, Vol. 1) located on the two strands in opposite orientations.

How do such relatively distant GATC sequences direct mismatch correction? The mechanism is illustrated in Fig. 25-23. MutL protein forms a complex with MutS protein, and this complex binds to all abnormal base pairs (except C-C). MutH protein binds to MutL and to the GATC sequences encountered by the MutL-MutS complex. DNA on both sides of the mismatched pair is pulled through the MutL-MutS complex, creating a loop. The simultaneous movement of both ends of the loop through the complex is equivalent to the complex moving along the DNA in both directions simultaneously. MutH protein possesses site-specific endonuclease activity, which remains latent until the complex encounters a hemimethylated GATC sequence. At this site, MutH catalyzes Cleavage of the unmethylated strand at the 5' side of the G residue within the GATC sequence, marking the strand for subsequent repair. Subsequent Stages of the pathway depend on the exact position of the mismatch relative to the cleavage site (Fig. 25-24).

Fig. 25-23. A model for the Cytology/cytology/16.html">Early stages of DNA Methylation-directed repair. The participating E. coli proteins (see Table 25-5) have been isolated and purified. Recognition of the 5’-(GATC) sequence and recognition of the mismatched base are the specific Functions of MutL and MutS proteins, respectively. MutH protein forms a complex with MutS at the site of the mismatch. DNA passes through this complex such that the complex moves in both directions simultaneously along the DNA until it contacts MutH bound to the hemimethylated GATC sequence. MutH protein cleaves the unmethylated strand on the 5' side of the guanine residue in this sequence. Then, a complex comprising DNA helicase II and an exonuclease cleaves the unmethylated strand from this point to the mismatched base (see Fig. 25-24).

Fig. 25-24. Completion of DNA methylation-based repair. Acting in concert, DNA helicase II, SSB, and one of four exonucleases remove a segment of the new strand between the site cleaved by the MutH protein and a point located just beyond the mismatched base. The choice of exonuclease depends on THE POSITION OF the cleavage site relative to the anomalous base pair. Alternative pathways are shown in the diagram. The resulting gap is filled by DNA polymerase III (dashed line), and the nick is sealed by DNA ligase (not shown).

If the mismatched base is located on the 5' side of the cleavage site (Fig. 25-24, right), the unmethylated strand is unwound and degraded in the 3' —> 5' direction from the cleavage site, encompassing the mismatched base, and this segment is replaced with new DNA. This process requires the coordinated action of DNA helicase II, SSB, exonuclease I or exonuclease X (both degrade DNA strands in the 3' —> 5' direction), DNA polymerase III, and DNA ligase. The mismatch repair pathway for mismatches located on the 3' side of the cleavage site differs only in that it involves exonuclease VII (which degrades single-stranded DNA in the 5' —> 3' or 3' —> 5' direction) or RecJ nuclease (which degrades single-stranded DNA in the 5' —> 3' direction).

Estimating the energy cost of single-nucleotide substitution repair in E. coli reveals that this is a highly resource-intensive process. An anomalous base pair can be located 1,000 bp or more away from GATC. Cleavage and resynthesis of a segment of this length require a huge number of activated deoxynucleotide precursors to repair a single nucleotide substitution. This once again underscores the critical importance of genome integrity for the cell.

All Eukaryotic cells contain several proteins that are structurally and functionally analogous to the bacterial proteins MutS and MutL (but not MutH). In humans, alterations in genes encoding this type of protein cause some of the most common inherited cancer predisposition syndromes (Box 25-1), further demonstrating the physiological importance of repair systems. In most eukaryotes, ranging from Yeast to humans,

the principal MutS homologs are the MSH2 (MutS homolog 2), MSH3, and MSH6 proteins. MSH2-MSH6 heterodimers typically bind to single anomalous base pairs, but show lower affinity for slightly longer mismatched loops. In many organisms, longer mismatch regions (2 to 6 bp) are bound by MSH2/MSH3 heterodimers or by Two Types of heterodimers in tandem. MutL homologs, particularly the MLH1 and PMS1 (postmeiotic segregation) heterodimer, associate with MSH complexes and stabilize them. Detailed studies of eukaryotic mismatch repair are actively ongoing. In particular, the mechanism by which newly synthesized DNA strands are identified remains unknown, although it has been established that GATC sequences are not involved in this process.

Base Excision Repair.

Every cell contains a class of Enzymes called DNA glycosylases; they recognize the most common DNA Lesions (e.g., cytosine and adenine deamination products; see Fig. 8-30a) and remove the damaged bases by cleaving the N-glycosidic bond. Such base excision generates an apurinic or apyrimidinic site in the DNA, referred to as an AP site or abasic site. Typically, each DNA glycosylase is specific for a single type of lesion.

Thus, uracil-DNA glycosylase, found in most cells, specifically removes from DNA the uracil generated by the Spontaneous deamination of cytosine. Mutant cells lacking this enzyme exhibit an increased mutation rate in G = C pairs compared to A = T pairs. This glycosylase does not remove

uracil residues from RNA or thymine residues from DNA. The need to discriminate between thymine and uracil (the deamination product of cytosine) for selective cytosine repair may be one of the reasons why DNA incorporates thymine rather than uracil (see p. 416, Vol. 1).

Most bacteria possess only a single type of uracil-DNA glycosylase, whereas humans have at least four types with distinct specificities, highlighting the importance of removing uracil from DNA. The most abundant human uracil glycosylase, UNG, associates with the replisome and excises uracil residues accidentally incorporated in place of thymine during replication. The deamination of cytosine residues occurs 100 times faster in single-stranded DNA than in double-stranded DNA, and humans possess a specialized enzyme, hSMUG1, that removes any uracil residues in single-stranded DNA during replication or Transcription. Two other human DNA glycosylases, TDG and MBD4, remove uracil and thymine residues paired with guanine, generated by the deamination of cytosine or 5-methylcytosine, respectively.

Other DNA glycosylases recognize and remove various damaged bases, including formamidopyrimidine and 8-hydroxyguanine (both formed by purine oxidation), hypoxanthine (formed by adenine deamination), and alkylated bases such as 3-methyladenine and 7-methylguanine. Glycosylases recognizing other defects, including pyrimidine dimers, have also been identified in certain classes of organisms. Recall that AP sites are also formed as a result of the slow spontaneous Hydrolysis of N-glycosidic bonds in DNA (see Fig. 8-30b, Vol. 1).

The AP site generated by DNA glycosylase is processed by Other Enzymes. Repair does not simply involve attaching a new base and restoring the N-glycosidic bond. Instead, the remaining deoxyribose-5'-phosphate is removed and a new nucleotide is inserted. This process is initiated by one of the AP endonucleases, which cleave the DNA strand containing the AP site. The position of the cut relative to the AP site (on the 3' or 5' side) depends on the type of AP endonuclease. Subsequently, the DNA segment encompassing the AP site is removed, DNA polymerase I synthesizes the replacement DNA, and DNA ligase seals the remaining nick (Fig. 25-25). In eukaryotes, nucleotide replacement is carried out by specialized polymerases, as described below.

Fig. 25-25. Base excision repair. ① A DNA glycosylase recognizes a damaged base and cleaves it from the deoxyribose in the DNA strand. ② An AP endonuclease cleaves the phosphodiester bond near the AP site. ③ DNA polymerase I initiates repair synthesis starting from the free 3'-hydroxyl group at the nick, simultaneously removing (via 5' —> 3' exonuclease activity) a portion of the damaged strand and replacing it with new DNA. ④ The nick remaining after the dissociation of DNA polymerase I is sealed by DNA ligase.

Nucleotide excision repair.

DNA lesions that cause significant distortions of the helical structure are typically repaired by the nucleotide excision repair system. This repair pathway is essential for the survival of all free-living organisms. In this repair system (Fig. 25-26), a multisubunit enzyme hydrolyzes two phosphodiester bonds, one on each side of the lesion. In E. coli and other bacteria, the enzyme system hydrolyzes the 5th phosphodiester bond on the 3' side and the 8th phosphodiester bond on the 5' side, releasing a 12–13 nucleotide fragment (depending on whether one or two bases are damaged). In humans and other eukaryotes, the enzyme system hydrolyzes the 6th phosphodiester bond on the 3' side and the 22nd phosphodiester bond on the 5' side, generating a 27–29 nucleotide fragment. As a result of this dual incision, the oligonucleotide containing the damaged region is released from the duplex, and the resulting gap is filled by DNA polymerase I in E. coli and DNA polymerase ε in humans. The nick is sealed by DNA ligase.

Fig. 25-26. Nucleotide excision repair in E. coli and humans. The core pathway of nucleotide excision repair is nearly identical across all organisms. ① An excision nuclease binds to the DNA at the site of a major lesion and cleaves the damaged DNA strand on both sides of the lesion. ② A 13-nucleotide (13-mer) or 29-nucleotide (29-mer) DNA fragment is removed by a helicase. ③ The gap is filled by a DNA polymerase, and ④ the remaining nick is sealed by DNA ligase.

In E. coli, this process is orchestrated by the ABC excision nuclease, which consists of three subunits: UvrA (M_r = 104,000), UvrB (M_r = 78,000), and UvrC (M_r = 68,000). The term "excision nuclease" describes The unique ability of this enzyme complex to catalyze dual specific cleavage, distinguishing it from conventional endonucleases. The UvrA and UvrB protein complex (A₂B) scans the DNA and binds at the lesion site. The UvrA dimer then dissociates, leaving a stable UvrB-DNA complex. Next, the UvrC protein binds to UvrB, and UvrB makes an incision at the 5th phosphodiester bond on the 3' side of the lesion. This is followed by a UvrC-mediated incision at the 8th phosphodiester bond on the 5' side. The resulting 12–13 nucleotide fragment is removed by DNA helicase. The resulting small gap is repaired by DNA polymerase I and DNA ligase. This is the primary repair pathway for many types of damage (see Fig. 25-26, left), including cyclobutane pyrimidine dimers, 6,4-photoproducts (see Fig. 8-31, Vol. 1), and various adducts, such as benzo[a]pyrene-guanine formed in DNA upon exposure to tobacco smoke. The nucleolytic activity of the ABC excision nuclease differs from that of other endonucleases in that it introduces two simultaneous incisions into the DNA.

The mechanism of eukaryotic excision Nucleases is remarkably similar to that of the bacterial enzyme, although dual excision in eukaryotic cells requires 16 Polypeptides that share no Sequence Homology with the subunits of the E. coli excision nuclease. As described in Chapter 26, nucleotide and base excision repair in eukaryotes are tightly coupled to transcription. Genetic Defects in the nucleotide excision repair system underlie various severe human diseases (see Box 25-1).

Direct Repair.

Certain types of damage are repaired without removing a nucleotide or base. The most characteristic example is the direct photoreactivation of cyclobutane pyrimidine dimers, which is carried out by DNA photolyases. Pyrimidine dimers are formed via a UV light-induced reaction, and photolyases harness the energy of absorbed light to reverse the damage (Fig. 25-27). Photolyases typically contain two Cofactors that function as light-absorbing agents (chromophores). One of the chromophores is always FADH. In E. coli and yeast, the second chromophore is a folate. Free radicals are generated during the reaction. Placental mammals (including humans) lack DNA photolyases.

Fig. 25-27. Reaction mechanism. Reversal of pyrimidine dimers by photolyase. The energy of absorbed light is used to undo the photoreaction that caused the lesion. In E. coli, two photolyase chromophores (Mr = 54,000) act in concert: N⁵, N¹⁰-methenyltetrahydrofolylpolyglutamate (MTHFpolyGlu) and FADH^-. MTHFpolyGlu acts as an antenna, absorbing blue light photons (300–500 nm). The excitation Energy is transferred to FADH^-, and the excited flavin (*FADH^-) transfers an electron to the pyrimidine dimer, repairing the damage.

Another example is the repair of nucleotides whose damage is caused by alkylation. The modified nucleotide O⁶-methylguanine is formed in the presence of alkylating agents; this is a widespread and highly mutagenic lesion (see p. 419, vol. 1). During replication, it pairs with thymine more readily than with cytosine, resulting in the substitution of a G = C pair for an A = T pair (Fig. 25-28). Direct repair of O⁶-methylguanine is carried out by O⁶-methylguanine-DNA methyltransferase, which catalyzes The transfer of the methyl group to one of its Cys residues. This methyltransferase is not a true enzyme, because as a result of a single Methyl group transfer, it remains methylated and is removed from the process. Consuming an entire protein molecule to fix a single damaged base is yet another striking illustration of the priority of cellular DNA integrity.

A different, though also direct, mechanism is involved in the repair of 1-methyladenine and 3-methylcytosine. The amino groups of A and C residues are sometimes methylated (usually in single-stranded DNA), which affects The fidelity of base pairing. In E. coli, the oxidative demethylation of such alkylated nucleotides is mediated by the AlkB protein, a member of the α-ketoglutarate-Fe²⁺-dependent dioxygenase superfamily (Fig. 25-29). (For a description of another enzyme from this group, see Box 4-3 in Vol. 1.)

Fig. 25-28. Example of mutation formation as a result of DNA damage. (a) The methylation product, O⁶-methylguanine, forms a base pair with thymine more readily than with cytosine. (b) In the absence of repair, this leads to a mutation: after replication, G = C is replaced by A = T.

Fig. 25-29. Direct repair of alkylated bases by the AlkB protein. The AlkB protein is an α-ketoglutarate-Fe²⁺-dependent dioxygenase; it catalyzes the oxidative demethylation of 1-methyladenine and 3-methylcytosine residues.

Interaction of replication forks with DNA damage can trigger error-prone translesion DNA Synthesis

The DNA repair pathways discussed above generally operate in cases of double-stranded DNA damage, where the intact strand ensures the accurate restoration of the genetic information of the damaged strand to its original state. However, with certain types of damage, such as double-strand breaks, cross-links, or single-stranded DNA damage, the complementary strand is also destroyed or missing. Double-strand breaks and single-stranded DNA damage most frequently occur when a replication fork encounters unrepaired DNA damage (Fig. 25-30). Such damage and DNA cross-links can also result from ionizing radiation and oxidative reactions.

Fig. 25-30. DNA damage and its effect on replication. If a replication fork encounters an uncorrected lesion or a broken strand, replication typically halts. Left: damage remaining in an unreplicated single-stranded DNA segment; right: a double-strand break. In either case, the damage in one strand cannot be repaired by the previously described mechanisms because the complementary strand required for accurate repair is damaged or absent. In such cases, There are two possible repair pathways: HOMOLOGOUS DNA RECOMBINATION repair (see Fig. 25-37) or, if the damage is too extensive, error-prone translesion synthesis. The latter mechanism involves a different DNA polymerase (DNA polymerase V, encoded by the umuC and umuD genes), which can replicate, albeit with errors, DNA regions containing various types of damage. This mechanism is called “error-prone” repair due to The high frequency of mutations it generates.

When a bacterial replication fork stalls, two repair pathways are possible. In the absence of the second strand, the information required for accurate repair must be provided by a homologous chromosome. Thus, the repair system utilizes homologous genetic recombination. Homologous DNA recombination repair is discussed in detail in Section 25.3. Under certain circumstances, a second repair pathway is implemented—error-prone translesion synthesis (TLS). Upon activation of this pathway, DNA repair becomes significantly less accurate and is associated with a high mutation frequency. In bacteria, error-prone translesion synthesis is part of the cell’s stress response to severe DNA damage, known as the SOS Response. Some SOS proteins, such as the previously described UvrA and UvrB (Table 25-6), are always present in the cell, but their levels increase significantly when the SOS response is triggered. Other SOS proteins also participate in error-prone repair, including UmuD and UmuC (“Umu” from immutable—non-mutable; in the absence of a functional gene, error-prone repair is absent). In the SOS-regulated process, the UmuD protein is cleaved into a shorter fragment, UmuD’, which forms a specialized DNA polymerase (DNA polymerase V) in complex with UmuC; this polymerase is capable of bypassing and repairing many DNA lesions that block replication. Correct base pairing is often impossible in the defective region, which is why such translesion replication is accompanied by errors.

Table 25–6. Genes Induced as Part of the SOS Response in E. coli

Some of these genes and their functions are discussed in Chapter 28.

Throughout this chapter, we have repeatedly emphasized the critical importance of genome preservation, and therefore the existence of a system that increases the frequency of mutations might seem counterintuitive. This system can be viewed as a desperate measure of last resort. The umuC and umuD genes are fully induced only in the late stages of the SOS response; they are not activated for synthesis—initiated by the cleavage of UmuD—until DNA damage becomes so extensive that all replication forks are stalled. The mutations arising from such replication lead to the death of some cells and pose risks to others, but this is the biological price the Organism pays to prevent the complete cessation of replication, thereby ensuring the survival of at least a few mutant daughter cells.

Along with DNA polymerase V, the RecA protein is required for translesion synthesis. RecA filaments bound to single-stranded DNA in one region of the chromosome can activate DNA polymerase V complexes bound at distant chromosomal sites. This so-called trans mechanism is facilitated by loop formation, which brings spatially distant chromosomal sequences into close proximity. Another DNA polymerase, DNA polymerase IV (the product of the dinB gene), is also induced during the SOS response. Replication catalyzed by DNA polymerase IV is likewise error-prone. Bacterial DNA polymerases IV and V belong to the TLS polymerase family found in all organisms. These enzymes lack proofreading 3'–5' exonuclease activity, and their base-Selection fidelity during replication is lower by a factor of 10², reducing overall replication accuracy to roughly one error per 1,000 nucleotides.

Mammals possess multiple low-fidelity TLS DNA polymerases. However, the presence of these enzymes does not inherently cause a high mutation frequency because most of them perform specialized repair functions. DNA polymerase η (eta), for example, is a TLS polymerase found in all eukaryotes. It carries out translesion synthesis primarily across cyclobutane T–T dimers. This process generates relatively few mutations because the enzyme preferentially inserts two A residues opposite the linked T residues. Eukaryotes have several polymerases, including DNA polymerases β, ι, and λ, that perform specialized, lower-fidelity functions in base excision repair. Each of these enzymes possesses 5'-deoxyribose phosphate lyase activity In addition to polymerase activity. Following base removal by a glycosylase and backbone cleavage by an AP endonuclease, these enzymes remove the AP site (5'-deoxyribose phosphate) and fill the very short gap. The mutation rate associated with DNA polymerase η remains low largely because it synthesizes very short stretches of DNA (often just a single nucleotide).

Studies of cellular DNA repair systems have revealed that DNA metabolism maintains genome integrity through multiple, often redundant pathways. The Human Genome contains over 130 genes encoding proteins associated with DNA repair. In many cases, the loss of a single gene's function leads to genome instability and an increased susceptibility to tumorigenesis (Box 25–1). These repair systems often operate in concert with DNA replication machinery and are complemented by recombination pathways, which are discussed below.

Box 25–1. MEDICINE. DNA Repair and Cancer

Human cancer arises when specific genes that regulate normal cell division (oncogenes and tumor suppressor genes; see Chapter 12, Vol. 1) malfunction, become altered, or are activated inappropriately. As a result, cell growth can spiral out of control, leading to a tumor. Genes that control cell division can be damaged by spontaneous mutations or disrupted by the insertion of tumor Viruses (Chapter 26). It is hardly surprising that defects in DNA repair genes, which lead to elevated mutagenesis, can drastically increase an individual's susceptibility to cancer. Defects in genes encoding proteins involved in nucleotide excision repair, mismatch repair, recombinational repair, and the SOS response are all linked to oncogenesis. Thus, life and death can literally depend on DNA repair.

Unlike in bacteria, nucleotide excision repair in humans requires a larger Complement of proteins, although the overall mechanisms are strikingly similar. Genetic defects that inactivate nucleotide excision repair are associated with several hereditary disorders, of which xeroderma pigmentosum (XP) is the best studied. Because nucleotide excision repair is the sole pathway for removing pyrimidine dimers in humans, individuals with XP are extremely sensitive to sunlight and frequently develop sunlight-induced Skin cancers. Most XP patients also exhibit neurological disorders, presumably due to their inability to repair specific lesions generated by the high levels of oxidative Metabolism in Neurons. Defects in genes encoding any of at least seven distinct protein Components of the nucleotide excision repair system give rise to seven complementation groups of XP (from XPA to XPG). Several of these proteins (particularly those defective in XPW, XPD, and XPG) also participate in transcription-coupled repair of oxidative damage, as described in Chapter 26.

Most microorganisms possess additional pathways to repair cyclobutane pyrimidine dimers—they can utilize DNA photolyase activity and, occasionally, alternative nucleotide excision repair mechanisms—but humans and other placental mammals lack this capability. The absence of alternatives for removing pyrimidine dimers via nucleotide excision repair has led to the hypothesis that early mammals were small, fur-covered, nocturnal animals with little need to repair UV-induced damage. Nevertheless, mammals possess A bypass pathway for cyclobutane pyrimidine dimers that involves DNA polymerase η. This enzyme preferentially inserts two A residues opposite a T–T pyrimidine dimer, minimizing the likelihood of mutation. Individuals who lack functional DNA polymerase η due to genetic mutations suffer from a disorder resembling xeroderma pigmentosum (XP variant, or XPV). The clinical manifestations of XPV mirror those of classical XP, although the mutation frequency in XPV cells is even higher upon UV irradiation. Apparently, in normal cells, the nucleotide excision repair system works cooperatively with DNA polymerase η to repair or bypass pyrimidine dimers, which is essential for normal cell growth and DNA replication. UV irradiation generates a multitude of pyrimidine dimers, and to sustain replication, at least some of these must be bypassed via translesion synthesis. If one system is absent, the other partially compensates. A deficiency in polymerase η activity stalls replication forks and forces UV-induced lesions to be bypassed by more error-prone TLS polymerases. In the absence of other DNA repair pathways, this surge in mutations frequently culminates in cancer.

One of the most common inherited hereditary cancer-predisposition syndromes is hereditary nonpolyposis Colorectal Cancer (HNPCC). This syndrome is caused by defects in DNA mismatch repair. The cells of humans and other eukaryotes contain several protein homologs of the bacterial MutL and MutS proteins (see Fig. 25–23). Defects in at least five different mismatch repair genes can elevate the risk of developing HNPCC. Mutations in hMLH1 (human homolog 1 of MutL) and hMSH2 (human homolog 2 of MutS) are particularly common. In individuals with HNPCC, cancer typically manifests at an early age, most frequently as colorectal cancer.

Most cases of breast cancer in women occur without any known genetic predisposition. However, about 10% of cases are linked to inherited defects in two genes, BRCA1 and BRCA2. In humans, the BRCA1 and BRCA2 proteins are exceptionally large (1,834 and 3,418 amino acid residues, respectively) and interact with numerous other proteins involved in transcription, chromosome maintenance, DNA repair, and Cell Cycle checkpoint control. Furthermore, the BRCA2 protein is intimately connected with the recombinational Repair of Double-strand breaks. Despite this, the exact molecular functions of BRCA1 and BRCA2 in these cellular processes remain to be fully elucidated. Women carrying defects in either the BRCA1 or BRCA2 gene face a lifetime breast cancer risk exceeding 80%.

Summary of Section 25.2 DNA Repair

■ Cells possess multiple DNA repair systems. In E. coli, mismatch repair operates by taking advantage of the transient lack of methylation at (5') GATC sequences in the newly synthesized strand.

■ Excision repair systems recognize and eliminate Damage caused by environmental factors (such as radiation and alkylating agents) as well as spontaneous nucleotide reactions. Some repair systems recognize and excise only damaged or incorrect bases, leaving an AP site (apurinic/apyrimidinic site) in the DNA, which is then excised and filled with new DNA through the action of other enzymes.

■ Nucleotide excision repair systems recognize and remove various bulky lesions and pyrimidine dimers. They excise a segment of the DNA strand containing the lesion, leaving a gap that is subsequently filled in by DNA polymerase and DNA ligase.

■ Certain types of DNA damage are reversed directly by reactions that undo the defect: pyrimidine dimers are converted back to monomeric Pyrimidines by photolyase, whereas the methyl group of O⁶-methylguanine is removed by a methyltransferase.

■ Severe damage to bacterial DNA triggers translesion synthesis (TLS) across the lesion, mediated by error-prone TLS polymerases. Eukaryotes have similar polymerases that perform specialized functions in DNA repair while minimizing the mutation rate.