Influence of template strandedness on in vitro replication of mutagen-damaged DNA.
We analyzed the ability of DNA polymerases to bypass damage on single- and double-stranded templates. In vitro DNA synthesis was studied on UV-irradiated and polyaromatic hydrocarbon reacted (benzo[a]pyrenediol epoxide and oxiranylpyrene) double-stranded templates by a protocol involving initiation on a uniquely nicked circular double-stranded template. The template was prepared by treating single-stranded (+)M13mp2 circular strands with mutagen and then hybridizing with restricted M13 RFmp2, followed by isolation of the nicked RFII forms. The protocol permits either (+), (-), or both strands to carry lesions. We found that the rules for termination and bypass of lesions previously observed with single-stranded DNA templates also hold for double-stranded templates. Termination of synthesis occurs primarily one nucleotide 3' to the lesion in the template strand. Bypass of UV-induced lesions can be followed in a series of three partial reactions in the presence of Mn2+ and dGMP, which relax the specificity of nucleotide insertion and 3'----5' exonuclease activity, respectively. There is no evidence for greater permissivity of bypass in double-as opposed to single-stranded templates. As with single-stranded templates, purines and preferentially deoxyadenosine (dA) are inserted opposite lesions. Lesions in the nontemplate strand elicit neither termination nor pausing. The addition of Rec A protein resulted in a measurable increase of bypass in this system.
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000494:pharmacology*; D001483:Base Sequence; D004249:DNA Damage; D004261:DNA Replication / Q000187:drug effects / Q000528:radiation effects; D004262:DNA Restriction Enzymes; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004101:Dihydroxydihydrobenzopyrenes / Q000494:pharmacology*; D009153:Mutagens; D011721:Pyrenes / Q000494:pharmacology*; D013698:Templates, Genetic; D014466:Ultraviolet Rays
D004101:Dihydroxydihydrobenzopyrenes; D009153:Mutagens; D011721:Pyrenes; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; C043956:1-oxiranylpyrene; D004259:DNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes