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(PP345) Clustered damage: 5,6-dihydrothymine retards base excision repair of an AP site by xrs5 nuclear extracts.
Byrne, Shaun1, Bellon, Sophie1, Lomax, Martine1, O'Neill, Peter*,1, 1 DNA Damage Group, Harwell, Oxon, UK
ABSTRACT- Clustered DNA damage sites (2 or more lesions within one or 2 helical turns of the DNA) are induced by ionising radiation and challenge the repair mechanisms of the cell. The processing of clustered lesions is influenced by the types of lesions and their relative positions to each other within the cluster. Simple bistranded clustered sites, containing an abasic site (AP) or HAP1-SSB on one strand and either 8-oxoG or 5,6-dihydrothymine (DHT), 1 or 5 bases 3' or 5' to each other, were synthesised in oligonucleotides and the repair carried out in mammalian nuclear extracts. Previous work has shown that lesions within a bistranded clustered DNA damage site are recognised less efficiently by base excision repair (BER) proteins than when present as single lesions. The clustered damage sites were repaired by incubation with xrs5 nuclear extracts. The repair of the AP site is retarded when DHT is placed at any of the tested positions 3' to the AP site on the opposing strand whereas the rejoining is not retarded when DHT is positioned to the 5' side relative to the AP site, when only short patch (SP) BER occurs. The major effect is reduction in the efficiency of the DNA ligase when the base lesion is present 3' to the AP site and that in the 3' orientation 2 or more bases may be inserted during long patch repair (LPR) repair of the AP site. With a HAP1-SSB, the retardation in the presence of DHT is greater than that seen with an AP site but involves similar pathways. From comparison with the findings with 8-oxoG, the dynamics and the contribution of long patch repair is dependent upon the composition of the cluster. To test if SP BER of clustered damage occurs if LPR pathway is suppressed, dATP, the second base to be inserted by DNA polymerase, was substituted with (ddATP). Introduction of ddATP into the repair gap effectively terminates BER by LPR but not short patch BER. Under these conditions surprisingly, a significant fraction of the AP sites (or HAP1-SSB) in the presence of either DHT or 8-oxoG were not repaired by SP BER even though LPR had been suppressed. Inefficient repair of AP sites/HAP1-SSB within clustered DNA damage sites prolongs the lifetime of the lesion and may increase the number of unrepaired AP sites/HAP1-SSB present at replication, leading to mutagenic events. *funded in part by EU Marie Curie Research Training Network (MRCT-2003-505086)
Key words: Clustered damage, base excision repair, dihydrothymine, abasic site
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