Novel Enzymatic Function of DNA Polymerase ν in Translesion DNA Synthesis Past Major Groove DNA−Peptide and DNA−DNA Cross-Links

Abstract

DNA polymerase ν (POLN or pol ν) is a newly discovered A family polymerase that generates a high error rate when incorporating nucleotides opposite dG; its translesion DNA synthesis (TLS) capability has only been demonstrated for high fidelity replication bypass of thymine glycol lesions. In the current investigation, we describe a novel TLS substrate specificity of pol ν, demonstrating that it is able to bypass exceptionally large DNA lesions whose linkages are through the DNA major groove. Specifically, pol ν catalyzed efficient and high fidelity TLS past peptides linked to N6-dA via a reduced Schiff base linkage with a γ-hydroxypropano-dA. Additionally, pol ν could bypass DNA interstrand cross-links with linkage between N6-dAs in complementary DNA strands. However, the chemically identical DNA−peptide and DNA interstrand cross-links completely blocked pol ν when they were located in the minor groove via a N2-dG linkage. Furthermore, we showed that pol ν incorporated a nucleotide opposite the 1,N6-etheno-dA (εdA) in an error-free manner and (+)-trans-anti-benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide-dA [(+)-BPDE-dA] in an error-prone manner, albeit with a greatly reduced capability. Collectively, these data suggest that although pol ν bypass capacity cannot be generalized to all major groove DNA adducts, this polymerase could be involved in TLS when genomic replication is blocked by extremely large major groove DNA lesions. In view of the recent observation that pol ν may have a role in cellular tolerance to DNA cross-linking agents, our findings provide biochemical evidence for the potential functioning of this polymerase in the bypass of some DNA−protein and DNA−DNA cross-links.

Error-prone Translesion Synthesis Past DNA-Peptide Cross-links Conjugated to the Major Groove of DNA via C5 of Thymidine

“…Furthermore, proteasomal inhibitors have been reported to slow down the repair of formaldehyde-induced DPCs in cells (62) and to interfere with intracellular repair of DPC-containing plasmids (58). In addition, several in vitro replication studies using site-specific substrates containing small peptides conjugated to the major or minor groove of DNA have provided direct evidence for the ability of lesion bypass polymerases to catalyze nucleotide incorporation opposite the DNA-peptide lesions (25)(26)(27). Huang et al (63) reported NMR structure of the KWKK peptide conjugated to the N 2 position of guanine, which was shown to orient in the minor groove.…”

“…Previous investigations have revealed that the ability of DNA polymerases to bypass DNA-peptide conjugates is dependent on the lesion size, the attachment site within the DNA, and polymerase identity (10,(21)(22)(23)(24)(25)(26)(27)(28)(29). Lloyd and co-workers (26) have reported that human polymerase (hpol) and its Escherichia coli orthologue pol IV were able to catalyze error-free primer extension past DNA templates containing tetra-and dodecapeptides conjugated to the N 2 position of guanine via a trimethylene linker.…”

“…Lloyd and co-workers (26) have reported that human polymerase (hpol) and its Escherichia coli orthologue pol IV were able to catalyze error-free primer extension past DNA templates containing tetra-and dodecapeptides conjugated to the N 2 position of guanine via a trimethylene linker. hpol was capable of bypassing both 4-and 12-mer peptides conjugated to N 6 of adenine via a trimethylene linkage, with correct base (dT) inserted opposite the adduct (27). In contrast, analogous adducts at the N 2 of guanine completely blocked DNA replication (27).…”

“…hpol was capable of bypassing both 4-and 12-mer peptides conjugated to N 6 of adenine via a trimethylene linkage, with correct base (dT) inserted opposite the adduct (27). In contrast, analogous adducts at the N 2 of guanine completely blocked DNA replication (27). Yamanaka et al (27) proposed that small major groove DPC adducts have sufficient conformational flexibility to be accommodated within the active site of TLS polymerases without disturbing primer-template-enzyme interactions, although the corresponding minor groove adducts block replication.…”

“…In contrast, analogous adducts at the N 2 of guanine completely blocked DNA replication (27). Yamanaka et al (27) proposed that small major groove DPC adducts have sufficient conformational flexibility to be accommodated within the active site of TLS polymerases without disturbing primer-template-enzyme interactions, although the corresponding minor groove adducts block replication. More recently, Guengerich and co-workers (29) reported that human polymerases and , as well as bacterial polymerases pol T7 and DPO4, were capable of replicating DNA containing S- [4-(N 6 -deoxyadenosinyl)-2,3-dihydroxybutyl] glutathione adducts (N 6 -dA-(OH) 2 butyl-GlyCys-␥Glu).…”