title: Repair of tandem base lesions in DNA by human cell extracts generates persisting single-strand breaks.
abstract: Clustered DNA damage, where two or more lesions are located proximal to each other on the same or opposite DNA strands, is frequently produced as a result of exposure to ionising radiation. It has been suggested that such complex damaged sites pose problems for repair pathways. In this study, we addressed the question of how two 8-oxoguanine lesions, located two nucleotides apart on the same DNA strand, are repaired. We find that in human cell extracts repair of either of the 8-oxoguanine lesions within a tandem damaged site is initiated randomly and that the majority of the initiated repair proceeds to completion. However, a fraction of the initiated repair is delayed at the stage of an incised AP site and the rate of further processing of this incised AP site is dependent on the position of the remaining 8-oxoguanine. If the remaining 8-oxoguanine residue is located near the 5' terminus of the incised abasic site, repair continues as efficiently as repair of a single 8-oxoguanine residue. However, repair is delayed after the incision step when the remaining 8-oxoguanine residue is located near the 3' terminus. Although the presence of the 8-oxoguanine residue near the 3' terminus did not affect either DNA polymerase beta activity or poly(ADP)ribose polymerase-1 affinity and turnover on an incised AP site, we find that 8-oxoguanine-DNA glycosylase has reduced ability to remove an 8-oxoguanine residue located near the 3' terminus of the incised AP site. We find that binding of the 8-oxoguanine-DNA glycosylase to this 8-oxoguanine residue inhibits DNA repair synthesis by DNA polymerase beta, thus delaying repair. We propose that interference between a DNA glycosylase and DNA polymerase during the repair of tandem lesions may lead to accumulation of the intermediate products that contain persisting DNA strand breaks.
authors.affiliation: MRC Radiation and Genome Stability Unit, Harwell, Oxfordshire OX11 0RD, UK.
authors.forename: Helen
authors.initials: H
authors.lastname: Budworth
authors.forename: Gemma
authors.initials: G
authors.lastname: Matthewman
authors.forename: Peter
authors.initials: P
authors.lastname: O'Neill
authors.forename: Grigory L
authors.initials: GL
authors.lastname: Dianov
mesh_terms: D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D045647:DNA Glycosylases / Q000378:metabolism; D019951:DNA Polymerase beta / Q000378:metabolism; D004260:DNA Repair; D004305:Dose-Response Relationship, Drug; D006147:Guanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D013997:Time Factors
chemical_list: D009841:Oligonucleotides; C024829:8-hydroxyguanine; D006147:Guanine; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D019951:DNA Polymerase beta; D045647:DNA Glycosylases