DNA Replication:- The Interaction of Replication Forks with DNA Damage Can Lead to Error-Prone Translesion DNA Synthesis
The repair pathways considered to this point generally work only for lesions in double-stranded DNA, the un damaged strand providing the correct genetic information to restore the damaged strand to its original state. However, in certain types of lesions, such as double strand breaks, double-strand cross-links, or lesions in a single-stranded DNA, the complementary strand is itself damaged or is absent. Double-strand breaks and lesions in single-stranded DNA most often arise when a replication fork encounters an unrepaired DNA lesion (Fig. 1). Such lesions and DNA cross-links can also result from ionizing radiation and oxidative reactions. At a stalled bacterial replication fork, there are two avenues for repair. In the absence of a second strand, the information required for accurate repair must come from a separate, homologous chromosome. The repair system thus involves homologous genetic recombination. This recombinational DNA repair is considered in detail in Section 25.3. Under some conditions, a second repair pathway, error-prone translesion DNA synthesis (often abbreviated TLS), becomes available. When this pathway is active, DNA repair becomes significantly less accurate and a high mutation rate can result. In bacteria, error-prone translesion DNA synthesis is part of a cellular stress response to extensive DNA damage known, appropriately enough, as the SOS response. Some SOS proteins, such as the UvrA and UvrB proteins already described (Table 25–6), are normally present in the cell but are induced to higher levels as part of the SOS response. Additional SOS proteins participate in the pathway for error-prone repair; these include the UmuC and UmuD proteins (“Umu” from un mutable; lack of the umu gene function eliminates error-prone repair). The UmuD protein is cleaved in an SOS-regulated process to a shorter form called UmuD, which forms a complex with UmuC to create a specialized DNA polymerase (DNA polymerase V) that can replicate past many of the DNA lesions that would normally block replication. Proper base pairing is often impossible at the site of such a lesion, so this translesion replication is error-prone. Given the emphasis on the importance of genomic integrity throughout this chapter, the existence of a system that increases the rate of mutation may seem in congruous. However, we can think of this system as a desperation strategy. The umuC and umuD genes are fully induced only late in the SOS response, and they are not activated for translesion synthesis initiated by UmuD cleavage unless the levels of DNA damage are particularly high and all replication forks are blocked. The mutations resulting from DNA polymerase V mediated replication kill some cells and create deleterious mutations in others, but this is the biological price an organism pays to overcome an otherwise insurmountable barrier to replication, as it permits at least a few mutant cells to survive. In addition to DNA polymerase V, translesion replication requires the RecA protein, SSB, and some sub units derived from DNA polymerase III. Yet another DNA polymerase, DNA polymerase IV, is also induced during the SOS response. Replication by DNA polymerase IV, a product of the dinBgene, is also highly error-prone. The bacterial DNA polymerases IV and V are part of a family of TLS polymerases found in all organisms. These enzymes lack a proofreading exonuclease activity, and the fidelity of replicative base selection can be reduced by a factor of 102, lowering overall replication fidelity to one error in ~1,000 nucleotides. Mammals have many low-fidelity DNA polymerases of the TLS polymerase family. However, the presence of these enzymes does not necessarily translate into an unacceptable mutational burden, because most of the enzymes also have specialized functions in DNA repair. DNA polymerase (eta), for example, is a TLS polymerase found in all eukaryotes. It promotes translesion synthesis primarily across cyclobutane T–T dimers. Few mutations result in this case, because the enzyme preferentially inserts two A residues across from the linked T residues. Several other low-fidelity polymerases, in cluding DNA polymerases, (iota), and, have specialized roles in eukaryotic base-excision repair. Each of these enzymes has a 5-deoxyribose phosphate lyase activity in addition to its polymerase activity. After base removal by a glycosylase and backbone cleavage by an AP endonuclease, these enzymes remove the abasicsite (a 5-deoxyribose phosphate) and fill in the very short gap with their polymerase activity. The frequency of mutations due to DNA polymerase activity is minimized by the very short lengths (often one nucleotide) of DNA synthesized.
What emerges from research into cellular DNA re pair systems is a picture of a DNA metabolism that maintains genomic integrity with multiple and often redundant systems. In the human genome, more than 130 genes encode proteins dedicated to the repair of DNA. In many cases, the loss of function of one of these proteins results in genomic instability and an increased occurrence of oncogenesis (Box 25–1). These repair systems are often integrated with the DNA replication systems and are complemented by the recombination systems that we turn to next.
FIGURE 1 DNA damage and its effect on DNA replication. If the replication fork encounters an unrepaired lesion or strand break, replication generally halts and the fork may collapse. A lesion is left behind in an unreplicated, single-stranded segment of the DNA; a strand break becomes a double-strand break. In each case, the damage to one strand cannot be repaired by mechanisms described earlier in this chapter, because the complementary strand required to direct accurate repair is damaged or absent. There are two possible avenues for repair: recombinational DNA repair or, when lesions are unusually numerous, error-prone repair. The latter mechanism involves a novel DNA polymerase (DNA polymerase V, encoded by the umuC and umuD genes) that can replicate, albeit inaccurately, over many types of lesions. The repair mechanism is referred to as error-prone because mutations often result.
