Gap-directed translesion DNA synthesis of an abasic site on circular DNA templates by a human replication complex.
DNA polymerase ε (pol ε) is believed to be the leading strand replicase in eukaryotes whereas pols λ and β are thought 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). We have previously reported that human pols λ, β and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε. In the case of pol λ and β, this TLS requires the presence of a gap downstream from the product synthetized by the ε replicase. However, since these studies were conducted exclusively with a linear DNA template, we decided to test whether the structure of the template could influence the capacity of the pols ε, λ, β and η to perform TLS of an AP site. Therefore, we have investigated the replication of damaged "minicircle" DNA templates. In addition, replication of circular DNA requires, beyond DNA pols, the processivity clamp PCNA, the clamp loader replication factor C (RFC), and the accessory proteins replication protein A (RPA). Finally we have compared the capacity of unmodified versus monoubiquitinated PCNA in sustaining TLS by pols λ and η on a circular template. Our results indicate that in vitro gap-directed TLS synthesis by pols λ and β in the presence of pol ε, RPA and PCNA is unaffected by the structure of the DNA template. Moreover, monoubiquitination of PCNA does not affect TLS by pol λ while it appears to slightly stimulate TLS by pol η.
Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France; Université de Toulouse, Université Paul Sabatier, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France.
Giuseppe
G
Villani
Leibniz Institute for Age Research - Fritz Lipman Institute, Jena, Germany; Department of Biochemistry, University of Oulu, Oulu, Finland.
Igor
I
Shevelev
Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France; Université de Toulouse, Université Paul Sabatier, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France.
Eleonora
E
Orlando
Leibniz Institute for Age Research - Fritz Lipman Institute, Jena, Germany; Department of Biochemistry, University of Oulu, Oulu, Finland.
Helmut
H
Pospiech
Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland.
Juhani E
JE
Syvaoja
Biochemistry Group, Department of Oncology, Gray Institute for Radiation Oncology and Biology, Oxford, United Kingdom.
Enni
E
Markkanen
Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich-Irchel, Zürich, Switzerland.
Ulrich
U
Hubscher
Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France; Université de Toulouse, Université Paul Sabatier, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France.
Nicolas Tanguy
NT
Le Gac
D004257:DNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D004261:DNA Replication; D004270:DNA, Circular; D006801:Humans
D004270:DNA, Circular; D004257:DNA Polymerase II; D019951:DNA Polymerase beta