DNA Replication:- All Cells Have Multiple DNA Repair Systems
The number and diversity of repair systems reflect both the importance of DNA repair to cell survival and the diverse sources of DNA damage (Table 25–5). Some common types of lesions, such as pyrimidine dimers (see Fig. 8–34), can be repaired by several distinct systems. Many DNA repair processes also appear to be extraordinarily inefficient energetically—an exception to the pattern observed in the metabolic pathways, where every ATP is generally accounted for and used optimally. When the integrity of the genetic information is at stake, the amount of chemical energy invested in a repair process seems almost irrelevant. DNA repair is possible largely because the DNA molecule consists of two complementary strands. DNA damage in one strand can be removed and accurately replaced by using the undamaged complementary strand as a template. We consider here the principal types of repair systems, beginning with those that repair the rarenucleotide mismatches that are left behind by replication.
Mismatch Repair Correction of the rare mismatches left after replication in E. coli improves the overall fidelity of replication by an additional factor of 102to 103. The mismatches are nearly always corrected to reflect the information in the old (template) strand, so the repair system must somehow discriminate between the template and the newly synthesized strand. The cell accomplishes this by tagging the template DNA with methyl groups to distinguish it from newly synthesized strands. The mismatch repair system of E. coliincludes at least 12 protein components (Table 25–5) that function either in strand discrimination or in the repair process itself.
The strand discrimination mechanism has not been worked out for most bacteria or eukaryotes, but is well understood for E.coli and some closely related bacteria. In these prokaryotes, strand discrimination is based on the action of Dam methylase (Table 25–3), which, as you will recall, methylates DNA at the N6position of all adenines within (5) GATC sequences. Immediately after passage of the replication fork, there is a short period (a few seconds or minutes) during which the template strand is methylated but the newly synthesized strand is not (Fig. 1). The transient unmethylated state of GATC sequences in the newly synthesized strand permits the new strand to be distinguished from the template strand. Replication mismatches in the vicinity of a hemimethylated GATC sequence are then repaired according to the information in the methylated parent (template) strand. Tests in vitro show that if both strands are methylated at a GATC sequence, few mismatches are repaired; if neither strand is methylated, repair occurs but does not favor either strand. The cell’s methyl-directed mismatch repair system efficiently re pairs mismatches up to 1,000 bp from a hemimethylated GATC sequence. For many bacterial species, the mechanism of strand discrimination during mismatch repair has not been determined. How is the mismatch correction process directed by relatively distant GATC sequences? A mechanism is illustrated in Figure 2. MutL protein forms a com plex with MutS protein, and the complex binds to all mismatched base pairs (except C–C). MutH protein binds to MutL and to GATC sequences encountered by the MutL-MutS complex. DNA on both sides of the mis match is threaded through the MutL-MutS complex, creating a DNA loop; simultaneous movement of both legs of the loop through the complex is equivalent to the complex moving in both directions at once along the DNA. MutH has a site-specific endonuclease activity that is inactive until the complex encounters a hemimethylated GATC sequence. At this site, MutH catalyzes cleavage of the unmethylated strand on the 5 side of the G in GATC, which marks the strand for repair. Further steps in the pathway depend on where the mismatch is located relative to this cleavage site (Fig. 3). When the mismatch is on the 5 side of the cleavage site, the unmethylated strand is unwound and degraded in the 3→5 direction from the cleavage site through the mismatch, and this segment is replaced with new DNA. This process requires the combined action of DNA helicase II, SSB, exonuclease I or exonuclease X (both of which degrade strands of DNA in the 3→5 direction), DNA polymerase III, and DNA ligase. The pathway for repair of mismatches on the 3 side of the cleavage site is similar, except that the exonuclease is either 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). Mismatch repair is a particularly expensive process for E. coli in terms of energy expended. The mismatch may be 1,000 bp or more from the GATC sequence. The degradation and replacement of a strand segment of this length require an enormous investment in activated deoxynucleotide precursors to repair a single mismatched base. This again underscores the importance to the cell of genomic integrity. All eukaryotic cells have several proteins structurally and functionally analogous to the bacterial MutS and MutL (but not MutH) proteins. Alterations in hu man genes encoding proteins of this type produce some of the most common inherited cancer-susceptibility syndromes (Box 25–1), further demonstrating the value to the organism of DNA repair systems. The main MutS homologs in most eukaryotes, from yeast to humans, are MSH2 (MutS homolog 2), MSH3, and MSH6. Heterodimers of MSH2 and MSH6 generally bind to single base-pair mismatches, and bind less well to slightly longer mispaired loops. In many organisms the longer mismatches (2 to 6 bp) may be bound instead by a heterodimer of MSH2 and MSH3, or are bound by both types of heterodimers in tandem. Homologs of MutL, predominantly a heterodimer of MLH1 and PMS1 (post meiotic segregation), bind to and stabilize the MSH complexes. Many details of the subsequent events in eu karyotic mismatch repair remain to be worked out. In particular, we do not know the mechanism by which newly synthesized DNA strands are identified, although research has revealed that this strand identification does not involve GATC sequences.
FIGURE 1 Methylation and mismatch repair. Methylation of DNA strands can serve to distinguish parent (template) strands from newly synthesized strands in E.coli DNA, a function that is critical to mismatch repair (see Fig. 25–21). The methylation occurs at the N6of adenines in (5)GATC sequences. This sequence is a palindrome (see Fig. 8–20), present in opposite orientations on the two strands.
FIGURE 2 A model for the early steps of methyl-directed mis match repair. The proteins involved in this process in E. coli have been purified (see Table 25–5). Recognition of the sequence (5) GATC and of the mismatch are specialized functions of the MutH and MutS proteins, respectively. The MutL protein forms a complex with MutS at the mismatch. DNA is threaded through this complex such that the complex moves simultaneously in both directions along the DNA until it encounters a MutH protein bound at a hemimethylated GATC sequence. MutH cleaves the unmethylated strand on the 5 side of the G in this sequence. A complex consisting of DNA helicase II and one of several exonucleases then degrades the unmethylated DNA strand from that point toward the mismatch (see Fig. 25–22).
FIGURE 3 Completing methyl-directed mismatch repair. The combined action of DNA helicase II, SSB, and one of four different exonucleases removes a segment of the new strand between the MutH cleavage site and a point just beyond the mismatch. The exonuclease that is used depends on the location of the cleavage site relative to the mismatch. The resulting gap is filled in by DNA polymerase III, and the nick is sealed by DNA ligase (not shown).
Base-Excision Repair Every cell has a class of enzymes called DNA glycosylases that recognize particularly common DNA lesions and remove the affected base by cleaving the N-glycosyl bond. This cleavage creates an apurinic or apyrimidinic site in the DNA, commonly referred to as an AP site or abasicsite. Each DNA glycosylase is generally specific for one type of lesion. Uracil DNA glycosylases, for example, found in most cells, specifically remove from DNA the uracil that results from spontaneous deamination of cytosine. Mutant cells that lack this enzyme have a high rate of G≡C to AUT mutations. This glycosylase does not remove uracil residues from RNA or thymine residues from DNA. The capacity to distinguish thymine from uracil, the product of cytosine deamination—necessary for the selective re pair of the latter—may be one reason why DNA evolved to contain thymine instead of uracil . Bacteria generally have just one type of uracil DNA glycosylase, whereas humans have at least four types, with different specificities—an indicator of the importance of uracil removal from DNA. The most abundant human uracil glycosylase, UNG, is associated with the human replisome, where it eliminates the occasional U residue inserted in place of a T during replication. The deamination of C residues is 100-fold faster in single stranded DNA than in double-stranded DNA, and humans have the enzyme hSMUG1, which removes any U residues that occur in single-stranded DNA during replication or transcription. Two other human DNA glycosylases, TDG and MBD4, remove either U or T residues paired with G, generated by deamination of cytosine or 5-methylcytosine, respectively. Other DNA glycosylases recognize and remove a variety of damaged bases, including formamidopyrimidine and 8-hydroxyguanine (both arising from purine oxidation), hypoxanthine (arising from adenine deamination), and alkylated bases such as 3-methyladenine and 7-methylguanine. Glycosylases that recognize other lesions, including pyrimidine dimers, have also been identified in some classes of organisms. Remember that AP sites also arise from the slow, spontaneous hydrolysis of the N-glycosyl bonds in DNA . Once an AP site has formed, another group of en zymes must repair it. The repair is notmade by simply inserting a new base and re-forming the N-glycosyl bond. Instead, the deoxyribose 5-phosphate left behind is removed and replaced with a new nucleotide. This process begins with AP endonucleases, enzymes that cut the DNA strand containing the AP site. The position of the incision relative to the AP site (5 or 3 to the site) varies with the type of AP endonuclease. A seg ment of DNA including the AP site is then removed, DNA polymerase I replaces the DNA, and DNA ligase seals the remaining nick (Fig. 4). In eukaryotes, nu cleotide replacement is carried out by specialized polymerases, as described below.
FIGURE 4 DNA repair by the base-excision repair pathway. 1 A DNA glycosylase recognizes a damaged base and cleaves between the base and deoxyribose in the backbone. 2 An AP endonuclease cleaves the phosphodiester backbone near the AP site. 3 DNA polymerase I initiates repair synthesis from the free 3 hydroxyl at the nick, removing (with its 5n3 exonuclease activity) a portion of the damaged strand and replacing it with undamaged DNA. 4 The nick remaining after DNA polymerase I has dissociated is sealed by DNA ligase.
Nucleotide-Excision Repair DNA lesions that cause large distortions in the helical structure of DNA generally are repaired by the nucleotide-excision system, a repair pathway critical to the survival of all free-living organ isms. In nucleotide-excision repair (Fig. 5), a multisubunit enzyme hydrolyzes two phosphodiester bonds, one on either side of the distortion caused by the lesion. In E.coli and other prokaryotes, the enzyme system hydrolyzes the fifth phosphodiester bond on the 3 side and the eighth phosphodiester bond on the 5 side to generate a fragment of 12 to 13 nucleotides (depending on whether the lesion involves one or two bases). In hu mans and other eukaryotes, the enzyme system hydrolyzes the sixth phosphodiester bond on the 3 side and the twenty-second phosphodiester bond on the 5 side, producing a fragment of 27 to 29 nucleotides. Fol lowing the dual incision, the excised oligonucleotides are released from the duplex and the resulting gap is filled—by DNA polymerase I in E.coli and DNA polymerase in humans. DNA ligase seals the nick. In E. coli, the key enzymatic complex is the ABC excinuclease, which has three subunits, UvrA (Mr 104,000), UvrB (Mr78,000), and UvrC (Mr68,000). The term “excinuclease” is used to describe the unique capacity of this enzyme complex to catalyze two specific endonucleolytic cleavages, distinguishing this activity from that of standard endonucleases. A complex of the UvrA and UvrB proteins (A2B) scans the DNA and binds to the site of a lesion. The UvrA dimer then dissociates, leaving a tight UvrB-DNA complex. UvrC protein then binds to UvrB, and UvrB makes an incision at the fifth phosphodiester bond on the 3 side of the lesion. This is followed by a UvrC-mediated incision at the eighth phosphodiester bond on the 5 side. The resulting 12 to 13 nucleotide fragment is removed by UvrD helicase. The short gap thus created is filled in by DNA polymerase I and DNA ligase. This pathway is a primary repair route for many types of lesions, including cyclobutane pyrimidine dimers, 6-4 photoproducts (see Fig. 8–34), and several other types of base adducts including benzo[a]pyrene-guanine, which is formed in DNA by exposure to cigarette smoke. The nucleolytic activity of the ABC excinuclease is novel in the sense that two cuts are made in the DNA (Fig. 5). The mechanism of eukaryotic excinucleases is quite similar to that of the bacterial enzyme, although 16 poly peptides with no similarity to the E. coli excinuclease subunits are required for the dual incision. As described in Chapter 26, some of the nucleotide-excision repair and base-excision repair in eukaryotes is closely tied to transcription. Genetic deficiencies in nucleotide excision repair in humans give rise to a variety of serious diseases (Box 25–1).
FIGURE 5 Nucleotide-excision repair in E. coli and humans. The general pathway of nucleotide-excision repair is similar in all organisms. 1 An excinuclease binds to DNA at the site of a bulky lesion and cleaves the damaged DNA strand on either side of the lesion. 2 The DNA segment—of 13 nucleotides (13 mer) or 29 nucleotides (29 mer)—is removed with the aid of a helicase. 3 The gap is filled in by DNA polymerase, and 4 the remaining nick is sealed with DNA ligase.
MECHANISM FIGURE 6 Repair of pyrimidine dimers with photolyase. Energy derived from absorbed light is used to reverse the photoreaction that caused the lesion. The two chromophores in E. coli photolyase (Mr 54,000), N5, N10-methenyltetrahydrofolylpolygluta mate (MTHF poly Glu) and FADH-, perform complementary functions. On binding of photolyase to a pyrimidine dimer, repair proceeds as follows. 1 A blue-light photon (300 to 500 nm wavelength) is absorbed by the MTHF poly Glu, which functions as a photo antenna. 2 The excitation energy passes to FADH in the active site of the enzyme. 3 The excited flavin (*FADH-) donates an electron to the pyrimidine dimer (shown here in a simplified representation) to generate an unstable dimer radical. 4 Electronic rearrangement restores the monomeric pyrimidines, and 5 the electron is transferred back to the flavin radical to regenerate FADH-.
Direct Repair Several types of damage are repaired without removing a base or nucleotide. The best-characterized example is direct photoreactivation of cyclobutane pyrimidine dimers, a reaction promoted by DNA photolyases. Pyrimidine dimers result from an ultraviolet light–induced reaction, and photolyases use energy derived from absorbed light to reverse the damage (Fig. 6). Photolyases generally contain two cofactors that serve as light-absorbing agents, or chromophores. One of the chromophores is always FADH-. In E. coli and yeast, the other chromophore is a folate. The reaction mechanism entails the generation of free radicals. DNA photolyases are not present in the cells of placental mammals (which include humans).
FIGURE 7 Example of how DNA damage results in mutations. (a) The methylation product O6-methylguanine pairs with thymine rather than cytosine. (b) If not repaired, this leads to a G≡C to A=T mutation after replication.
Additional examples can be seen in the repair of nucleotides with alkylation damage. The modified nu cleotide O6-methylguanine forms in the presence of alkylating agents and is a common and highly mutagenic lesion . It tends to pair with thymine rather than cytosine during replication, and therefore causes G≡C to A=T mutations (Fig. 7). Direct repair of O6 methylguanine is carried out by O6-methylguanine-DNA methyltransferase, a protein that catalyzes transfer of the methyl group of O6-methylguanine to one of its own Cys residues. This methyltransferase is not strictly an enzyme, because a single methyl transfer event permanently methylates the protein, making it inactive in this pathway. The consumption of an entire protein molecule to correct a single damaged base is another vivid illustration of the priority given to maintaining the integrity of cellular DNA.
A very different but equally direct mechanism is used to repair 1-methyladenine and 3-methylcytosine. The amino groups of A and C residues are sometimes methylated when the DNA is single-stranded, and the methylation directly affects proper base pairing. In E. coli, oxidative demethylation of these alkylated nucleotides is mediated by the AlkB protein, a member of the -ketoglutarate-Fe2–dependent dioxygenase superfamily (Fig. 8). (See Box 4–3 for a description of another member of this enzyme family.)
