Dataset Information

Y-family DNA polymerase-independent gap-filling translesion synthesis across aristolochic acid-derived adenine adducts in mouse cells.

ABSTRACT: Translesion DNA synthesis (TLS) operates when replicative polymerases are blocked by DNA lesions. To investigate the mechanism of mammalian TLS, we employed a plasmid bearing a single 7-(deoxyadenosine-N6-yl)-aristolactam I (dA-AL-I) adduct, which is generated by the human carcinogen, aristolochic acid I, and genetically engineered mouse embryonic fibroblasts. This lesion induces A to T transversions at a high frequency. The simultaneous knockouts of the Polh, Poli and Polk genes did not influence the TLS efficiency or the coding property of dA-AL-I, indicating that an unknown DNA polymerase(s) can efficiently catalyze the insertion of a nucleotide opposite the adduct and subsequent extension. Similarly, knockout of the Rev1 gene did not significantly affect TLS. However, knockout of the Rev3l gene, coding for the catalytic subunit of pol?, drastically suppressed TLS and abolished dA-AL-I to T transversions. The results support the idea that Rev1 is not essential for the cellular TLS functions of pol? in mammalian cells. Furthermore, the frequency of dA-AL-I to T transversion was affected by a sequence context, suggesting that TLS, at least in part, contributes to the formation of mutational hot and cold spots observed in aristolochic acid-induced cancers.

SUBMITTER: Hashimoto K

PROVIDER: S-EPMC5048550 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Y-family DNA polymerase-independent gap-filling translesion synthesis across aristolochic acid-derived adenine adducts in mouse cells.

Hashimoto Keiji K Bonala Radha R Johnson Francis F Grollman Arthur P AP Moriya Masaaki M

DNA repair 20160729

Translesion DNA synthesis (TLS) operates when replicative polymerases are blocked by DNA lesions. To investigate the mechanism of mammalian TLS, we employed a plasmid bearing a single 7-(deoxyadenosine-N<sup>6</sup>-yl)-aristolactam I (dA-AL-I) adduct, which is generated by the human carcinogen, aristolochic acid I, and genetically engineered mouse embryonic fibroblasts. This lesion induces A to T transversions at a high frequency. The simultaneous knockouts of the Polh, Poli and Polk genes did ...[more]

PMID: 27497692

Similar Datasets

Project description:B-family DNA polymerases (PolBs) of different groups are widespread in Archaea, and different PolBs often coexist in the same organism. Many of these PolB enzymes remain to be investigated. One of the main groups that is poorly characterized is PolB2, whose members occur in many archaea but are predicted to be inactivated forms of DNA polymerase. Here, Sulfolobus islandicus DNA polymerase 2 (Dpo2), a PolB2 enzyme, was expressed in its native host and purified. Characterization of the purified enzyme revealed that the polymerase possesses a robust nucleotide incorporation activity but is devoid of the 3'-5' exonuclease activity. Enzyme kinetics analyses showed that Dpo2 replicates undamaged DNA templates with high fidelity, which is consistent with its inefficient nucleotide insertion activity opposite different DNA lesions. Strikingly, the polymerase is highly efficient in extending mismatches and mispaired primer termini once a nucleotide is placed opposite a damaged site. This extender polymerase represents a novel type of prokaryotic PolB specialized for DNA damage repair in Archaea. IMPORTANCE In this work, we report that Sulfolobus islandicus Dpo2, a B-family DNA polymerase once predicted to be an inactive form, is a bona fide DNA polymerase functioning in translesion synthesis. S. islandicus Dpo2 is a member of a large group of B-family DNA polymerases (PolB2) that are present in many archaea and some bacteria, and they carry variations in well-conserved amino acids in the functional domains responsible for polymerization and proofreading. However, we found that this prokaryotic B-family DNA polymerase not only replicates undamaged DNA with high fidelity but also extends mismatch and DNA lesion-containing substrates with high efficiencies. With these data, we propose this enzyme functions as an extender polymerase, the first prokaryotic enzyme of this type. Our data also suggest this PolB2 enzyme represents a functional counterpart of the eukaryotic DNA polymerase Pol zeta, an enzyme that is devoted to DNA damage repair.

Dataset Information

Y-family DNA polymerase-independent gap-filling translesion synthesis across aristolochic acid-derived adenine adducts in mouse cells.

Publications

Y-family DNA polymerase-independent gap-filling translesion synthesis across aristolochic acid-derived adenine adducts in mouse cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets