title: Tolerance for 8-oxoguanine but not thymine glycol in alignment-based gap filling of partially complementary double-strand break ends by DNA polymerase lambda in human nuclear extracts.
abstract: Ionizing radiation induces various clustered DNA lesions, including double-strand breaks (DSBs) accompanied by nearby oxidative base damage. Previous work showed that, in HeLa nuclear extracts, DSBs with partially complementary 3' overhangs and a one-base gap in each strand are accurately rejoined, with the gaps being filled by DNA polymerase lambda. To determine the possible effect of oxidative base damage on this process, plasmid substrates were constructed containing overhangs with 8-oxoguanine or thymine glycol in base-pairing positions of 3-base (-ACG or -GTA) 3' overhangs. In this context, 8-oxoguanine was well tolerated by the end-joining machinery when present at one end of the break, but not when present at both ends. Thymine glycol was less well tolerated than 8-oxoguanine, reducing gap filling and accurate rejoining by at least 10-fold. The results suggest that complex DSBs can be accurately rejoined despite the presence of accompanying base damage, but that nonplanar bases constitute a major barrier to this process and promote error-prone joining. A chimeric DNA polymerase, in which the catalytic domain of polymerase lambda was replaced with that of polymerase beta, could not substitute for polymerase lambda in these assays, suggesting that this domain is specifically adapted for gap filling on aligned DSB ends.
authors.affiliation: Department of Pharmacology and Toxicology and Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Rui-Zhe
authors.initials: RZ
authors.lastname: Zhou
authors.forename: Luis
authors.initials: L
authors.lastname: Blanco
authors.forename: Miguel
authors.initials: M
authors.lastname: Garcia-Diaz
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Bebenek
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Kunkel
authors.forename: Lawrence F
authors.initials: LF
authors.lastname: Povirk
mesh_terms: D020137:Base Pair Mismatch; D002457:Cell Extracts; D002467:Cell Nucleus / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D019951:DNA Polymerase beta / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013941:Thymine / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: D002457:Cell Extracts; D011993:Recombinant Fusion Proteins; C029389:thymine glycol; C024829:8-hydroxyguanine; D006147:Guanine; C415367:DNA polymerase beta2; D019951:DNA Polymerase beta; D013941:Thymine