Loss of DNA minor groove interactions by exonuclease-deficient Klenow polymerase inhibits O6-methylguanine and abasic site translesion synthesis.
The importance of DNA polymerase-DNA minor groove interactions on translesion synthesis (TLS) was examined in vitro using variants of exonuclease-deficient Klenow polymerase and site-specifically modified DNA oligonucleotides. Polymerase variant R668A lacks primer strand interactions, while variant Q849A lacks template strand interactions. O(6)-Methylguanine (m6G) and abasic site TLS was examined in three stages: dNTP insertion opposite the lesion, extension from a terminal lesion-containing base pair, and the dissociation equilibrium of the polymerase from the lesion-containing template. Less than 5% TLS was observed at the insertion step for either variant on the lesion-containing templates. While extensive TLS was observed for WT polymerase on the m6G template, only incorporation opposite the lesion was observed for the R668A variant. Loss of the template strand interaction, Q849A, resulted in the inability to insert dNTPs opposite either the m6G or abasic lesion. For both variants, extension of purine-containing m6G primer-templates was increased relative to WT polymerase. We observed similar extension efficiencies for all variants, relative to WT, using abasic template-primers. Polymerase dissociation/reassociation was studied through the use of a competitor primer/template complex. Dissociation for WT polymerase increased 2-fold and 3-fold, respectively, for m6G and abasic lesion-containing templates, relative to the natural template. Variants lacking DNA minor groove interactions displayed increased dissociation from DNA templates, relative to WT polymerase, but do not display an increased level of lesion-induced polymerase dissociation. Our results indicate that the primer and template strand interactions of the Klenow polymerase with the DNA minor groove are critical for maintaining the DNA-polymerase complex during translesion synthesis.
Gestl
Erin E
EE
The Gittlen Cancer Research Institute, The Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, Pennsylvania 17033, USA.
Eckert
Kristin A
KA
D019943:Amino Acid Substitution / Q000235:genetics; D020137:Base Pair Mismatch / Q000235:genetics; D018736:DNA Adducts / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D004249:DNA Damage; D004256:DNA Polymerase I / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000378:metabolism; D004260:DNA Repair; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D005092:Exonucleases / Q000172:deficiency* / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000096:biosynthesis / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D013698:Templates, Genetic
D018736:DNA Adducts; D017931:DNA Primers; D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; D006147:Guanine; C008449:O-(6)-methylguanine; D004256:DNA Polymerase I; D005092:Exonucleases