Replication fork collapse is a major cause of the high mutation frequency at three-base lesion clusters.
Unresolved repair of clustered DNA lesions can lead to the formation of deleterious double strand breaks (DSB) or to mutation induction. Here, we investigated the outcome of clusters composed of base lesions for which base excision repair enzymes have different kinetics of excision/incision. We designed multiply damaged sites (MDS) composed of a rapidly excised uracil (U) and two oxidized bases, 5-hydroxyuracil (hU) and 8-oxoguanine (oG), excised more slowly. Plasmids harboring these U-oG/hU MDS-carrying duplexes were introduced into Escherichia coli cells either wild type or deficient for DNA n-glycosylases. Induction of DSB was estimated from plasmid survival and mutagenesis determined by sequencing of surviving clones. We show that a large majority of MDS is converted to DSB, whereas almost all surviving clones are mutated at hU. We demonstrate that mutagenesis at hU is correlated with excision of the U placed on the opposite strand. We propose that excision of U by Ung initiates the loss of U-oG-carrying strand, resulting in enhanced mutagenesis at the lesion present on the opposite strand. Our results highlight the importance of the kinetics of excision by base excision repair DNA n-glycosylases in the processing and fate of MDS and provide evidence for the role of strand loss/replication fork collapse during the processing of MDS on their mutational consequences.
Institut Curie, Centre de Recherche, F-91405 Orsay, France; CNRS UMR3348, F-91405 Orsay, France and CEA, Institut de Radiobiologie Cellulaire et Moléculaire, 18 route du Panorama, F-92265 Fontenay aux Roses, France.
Yuliya
Y
Sedletska
J Pablo
JP
Radicella
Evelyne
E
Sage
D002461:Cell Line, Transformed; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D016296:Mutagenesis; D059645:Mutation Rate; D014498:Uracil / Q000031:analogs & derivatives / Q000378:metabolism
D029968:Escherichia coli Proteins; C045975:5-hydroxyuracil; C024829:8-hydroxyguanine; D014498:Uracil; D006147:Guanine; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli