Quantitative Analysis of Translesion DNA Synthesis across a Benzo[a]pyrene-Guanine Adduct in Mammalian Cells

Abstract

Replication across unrepaired DNA lesions in mammalian cells is effected primarily by specialized, low fidelity DNA polymerases. We studied translesion DNA synthesis (TLS) across a benzo[a]pyrene-guanine (BP-G) adduct, a major mutagenic DNA lesion generated by tobacco smoke. This was done using a quantitative assay that measures TLS indirectly, by measuring the recovery of gapped plasmids transfected into cultured mammalian cells. Analysis of PolK ؉/؉ mouse embryo fibroblasts (MEFs) showed that TLS across the BP-G adduct occurred with an efficiency of 48 ؎ 4%, which is an order of magnitude higher than in Escherichia coli. In PolK ؊/؊ MEFs, bypass was 16 ؎ 1%, suggesting that at least twothirds of the BP-G adducts in MEFs were bypassed exclusively by polymerase (pol ). In contrast, pol was not required for bypass across BP-G in a human XP-V cell line. Analysis of misinsertion specificity across BP-G revealed that bypass was more error-prone in MEFs lacking pol . Expression of pol from a plasmid introduced into PolK ؊/؊ MEFs restored both the extent and fidelity of bypass across BP-G. Pol was not required for bypass of a synthetic abasic site. In vitro analysis demonstrated efficient bypass across BP-G by both pol and pol , suggesting that the biological role of pol in TLS across BP-G is due to regulation of TLS and not due to an exclusive ability to bypass this lesion. These results indicate that BP-G is bypassed in mammalian cells with relatively high efficiency and that pol bypasses BP-G in vivo with higher efficiency and higher accuracy than other DNA polymerases.Genomic DNA is constantly subject to damage caused by both external agents, such as sunlight, and endogenous chemicals, such as reactive oxygen species. Most of this damage is eliminated by error-free DNA repair mechanisms, thereby restoring the DNA to its native sequence (1). However, a significant number of lesions escape repair and might therefore interfere with DNA replication and gene expression. Such interference can be mitigated by DNA damage tolerance mechanisms, primarily translesion DNA synthesis (TLS 1 ; also termed translesion replication) (2-5) and postreplicative recombinational repair (1, 6 -8). The key components in TLS are low fidelity DNA polymerases that specialize in lesion bypass (9 -12). These proteins were conserved in evolution and are present in organisms ranging from Escherichia coli to humans (13). Humans contain at least four specialized DNA polymerases belonging to the Y superfamily (pol , pol , pol , and REV1) as well as several from other polymerase families (e.g. pol (14), pol (15, 16), and pol (16, 17)). Many of these polymerases have been implicated in TLS in vitro (18 -24). However, there is a paucity of information about the efficiency and fidelity with which they support lesion bypass in living cells.Pol has a well established biological role in TLS, since it is mutated in all patients examined with the variant form of the hereditary disease xeroderma pigmentosum (10,18). This disease is characterized by sensitivi...

Lesion processing: high-fidelity versus lesion-bypass DNA polymerases

“…The minor-groove lesion positioning is suggested to be relevant to other Y family DNA polymerases of the DinB branch, including the human homolog Pol κ [22]. The DinB polymerases seem to have a special role in bypassing N 2 -dG lesions [38], with higher efficiency and accuracy than other DNA polymerases in cellular [39] and in vitro environments [40,41].…”

Proficient and Accurate Bypass of Persistent DNA Lesions by DinB DNA Polymerases

“…Although deletion of either gene does not alter the frequency of spontaneous mutation, 7,17 it results in an increase in the frequency of mutagenesis induced by certain DNA damaging agents. 11,20 Such behavior is analogously observed with respect to UV-induced mutation in human cells deficient in pol h function, which replicates relatively accurately over thymine-thymine cyclobutane dimers. 30,31 Substrate specificity also appears to be conserved between DinB and pol k. Both enzymes are able to bypass N 2 -B[a]P-adducted template G. 20,32,33 Moreover, they each display a striking 10-15 fold increased activity on a template N 2 -furfuryl-dG relative to undamaged DNA.…”

“…11,20 Such behavior is analogously observed with respect to UV-induced mutation in human cells deficient in pol h function, which replicates relatively accurately over thymine-thymine cyclobutane dimers. 30,31 Substrate specificity also appears to be conserved between DinB and pol k. Both enzymes are able to bypass N 2 -B[a]P-adducted template G. 20,32,33 Moreover, they each display a striking 10-15 fold increased activity on a template N 2 -furfuryl-dG relative to undamaged DNA. 11 Increased proficiency is also displayed by pol h replicating its cognate substrate T-T cyclobutane pyrimidine dimers.…”

“…18 Pol k -/-mouse embryonic fibroblasts are sensitive to benzo[a]pyrene 19 and are impaired in replicating a gapped plasmid containing a site-specific N 2 -B[a]P-dG lesion. 20 Pol k -/-mice also show increased spontaneous mutagenesis 18 and pol k appears to play a role in recovery from a B[a]P-induced S-phase checkpoint. 21 Similarly to E. coli DinB, overproduction of pol k not only increases mutation frequency but also promotes double strand breaks (DSB), increased homologous recombination and nonhomologous end joining, loss of heterozygosity (LOH), and aneuploidy.…”

Translesion synthesis in mammalian cells

“…Many in vitro studies have been carried out using different damaged DNA substrates, and it has been concluded that some of the polymerases are more effective at inserting nucleotides across from damaged bases but are unable to extend from the inserted nucleotide (eg polι), whereas others are less efficient at this insertion step but can extend from a nucleotide inserted by another polymerase opposite a damaged base. Both in vivo and in vitro studies have shown that polκ can carry out TLS past DNA containing benzo[a]pyrene -guanine adducts [12,13].…”