DNA polymerase (pol) is thought to be the leading strand replicase in eukaryotes, whereas pols and are believed to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol is halted by an abasic site (apurinic/apyrimidinic (AP) site). In this study, we present in vitro evidence that human pols , , and can perform translesion synthesis (TLS) of an AP site in the presence of pol , likely by initiating the 3’OHs created at the lesion by the arrested pol . However, in the case of pols and , this TLS requires the presence of a DNA gap downstream from the product synthesized by the pol , 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 . Characterization of the reaction products showed that pol inserted dAMP opposite the AP site, whereas gap filling synthesis by pol resulted in single or double deletions opposite the lesion. The synthesis up to the AP site by pol and the subsequent TLS by pols and 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 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 and the repair pols and . DOI: https://doi.org/10.1074/jbc.M111.246611 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-56814 Journal Article Accepted Version Originally published at: Villani, G; Hübscher, U; Gironis, N; Parkkinen, S; Pospiech, H; Shevelev, I; di Cicco, G; Markkanen, E; Syväoja, J E; Tanguy Le Gac, N (2011). In vitro gap-directed translesion DNA synthesis of an abasic site involving human DNA polymerases epsilon, lambda, and beta. Journal of Biological Chemistry, 286(37):32094-32104. DOI: https://doi.org/10.1074/jbc.M111.246611 Tanguy Le Gac Markkanen, Juhani E. Syvaoja and Nicolas Igor Shevelev, Giulia di Cicco, Enni Gironis, Sinikka Parkkinen, Helmut Pospiech, Giuseppe Villani, Ulrich Hubscher, Nadege . β and λ DNA polymerases ?, synthesis of an abasic site involving human In vitro gap directed translesion DNA Regular Paper: published online July 13, 2011 J. Biol. Chem. 10.1074/jbc.M111.246611 Access the most updated version of this article at doi: . JBC Affinity Sites Find articles, minireviews, Reflections and Classics on similar topics on the