title: In vitro gap-directed translesion DNA synthesis of an abasic site involving human DNA polymerases epsilon, lambda, and beta.
abstract: DNA polymerase (pol) ε is thought to be the leading strand replicase in eukaryotes, whereas pols λ and β are believed to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). In this study, we present in vitro evidence that human pols λ, β, and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε, likely by initiating the 3'OHs created at the lesion by the arrested pol ε. However, in the case of pols λ and β, this TLS requires the presence of a DNA gap downstream from the product synthesized by the pol ε, and the optimal gap for efficient TLS is different for the two polymerases. The presence of gaps did not affect the TLS capacity of human pol η. Characterization of the reaction products showed that pol β inserted dAMP opposite the AP site, whereas gap filling synthesis by pol λ resulted in single or double deletions opposite the lesion. The synthesis up to the AP site by pol ε and the subsequent TLS by pols λ and β are not influenced by human processivity factor proliferating cell nuclear antigen and human single-stranded DNA-binding protein replication protein A. The bypass capacity of pol λ at the AP site is greatly reduced when a truncated form of the enzyme, which has lost the BRCA1 C-terminal and proline-rich domains, is used. Collectively, our in vitro results support the existence of a mechanism of gap-directed TLS at an AP site involving a switch between the replicative pol ε and the repair pols λ and β.
authors.affiliation: Institut de Pharmacologie et de Biologie Structurale, CNRS-Université Paul Sabatier Toulouse III, UMR 5089, 205 Route de Narbonne, 31077 Toulouse Cedex, France.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Villani
authors.forename: Ulrich
authors.initials: U
authors.lastname: Hubscher
authors.forename: Nadege
authors.initials: N
authors.lastname: Gironis
authors.forename: Sinikka
authors.initials: S
authors.lastname: Parkkinen
authors.forename: Helmut
authors.initials: H
authors.lastname: Pospiech
authors.forename: Igor
authors.initials: I
authors.lastname: Shevelev
authors.forename: Giulia
authors.initials: G
authors.lastname: di Cicco
authors.forename: Enni
authors.initials: E
authors.lastname: Markkanen
authors.forename: Juhani E
authors.initials: JE
authors.lastname: Syväoja
authors.forename: Nicolas
authors.initials: N
authors.lastname: Tanguy Le Gac
mesh_terms: D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004257:DNA Polymerase II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D004247:DNA; C415367:DNA polymerase beta2; D004257:DNA Polymerase II; D019951:DNA Polymerase beta