Translesion Synthesis across the N 2 -Ethyl-deoxyguanosine Adduct by Human PrimPol.
Elizaveta O BoldinovaPratibha P GhodkeSruthi SudhakarVipin Kumar MishraAnna A ManukyanNataliya MiropolskayaPushpangadan I PradeepkumarAlena V MakarovaPublished in: ACS chemical biology (2022)
Primase-DNA polymerase (PrimPol) is involved in reinitiating DNA synthesis at stalled replication forks. PrimPol also possesses DNA translesion (TLS) activity and bypasses several endogenous nonbulky DNA lesions in vitro. Little is known about the TLS activity of PrimPol across bulky carcinogenic adducts. We analyzed the DNA polymerase activity of human PrimPol on DNA templates with seven N 2 -dG lesions of different steric bulkiness. In the presence of Mg 2+ ions, bulky N 2 -isobutyl-dG, N 2 -benzyl-dG, N 2 -methyl(1-naphthyl)-dG, N 2 -methyl(9-anthracenyl)-dG, N 2 -methyl(1-pyrenyl)-dG, and N 2 -methyl(1,3-dimethoxyanthraquinone)-dG adducts fully blocked PrimPol activity. At the same time, PrimPol incorporated complementary deoxycytidine monophosphate (dCMP) opposite N 2 -ethyl-dG with moderate efficiency but did not extend DNA beyond the lesion. We also demonstrated that mutation of the Arg288 residue abrogated dCMP incorporation opposite the lesion in the presence of Mn 2+ ions. When Mn 2+ replaced Mg 2+ , PrimPol carried out DNA synthesis on all DNA templates with N 2 -dG adducts in standing start reactions with low efficiency and accuracy, possibly utilizing a lesion "skipping" mechanism. The TLS activity of PrimPol opposite N 2 -ethyl-dG but not bulkier adducts was stimulated by accessory proteins, polymerase delta-interacting protein 2 (PolDIP2), and replication protein A (RPA). Molecular dynamics studies demonstrated the absence of stable interactions with deoxycytidine triphosphate (dCTP), large reactions, and C1'-C1' distances for the N 2 -isobutyl-dG and N 2 -benzyl-dG PrimPol complexes, suggesting that the size of the adduct is a limiting factor for efficient TLS across minor groove adducts by PrimPol.