Evidence for in vitro translesion DNA synthesis past a site-specific aminofluorene adduct.
作者： M L Michaels , D L Johnson , T M Reid , C M King , L J Romano
DOI: 10.1016/S0021-9258(18)47845-3
关键词:
摘要: The ability of Escherichia coli DNA polymerase I and T7 to bypass bulky C-8 guanyl-2-aminofluorene adducts in was studied by vitro synthesis reactions on a site-specific aminofluorene-modified M13mp9 template. This site-specifically modified prepared ligating an oligonucleotide containing single aminofluorene adduct into gapped heteroduplex (Johnson, D. L., Reid, T. M., Lee, M.-S., King, C. Romano, L. J. (1986) Biochemistry 25, 449-456). resulting covalently closed duplex molecule then cleaved with restriction endonuclease, denatured, annealed primer the 3' side form template specifically designed study bypass. In this system, any that not blocked would proceed 5' terminus single-stranded template, while interrupted terminate at or near location. We have measured find amount radiolabeled nucleotide incorporated either E. (large fragment) much greater than be predicted if were absolute block synthesis. Furthermore, products similar electrophoresed polyacrylamide gels showed conclusively majority synthesized bypassed lesion. Substitution Mn2+ for Mg2+ as divalent cation resulted even higher levels translesion
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