Incorporation and excision of 9-(2-phosphonylmethoxyethyl)guanine (PMEG) by DNA polymerase delta and epsilon in vitro.
PMEG (9-(2-phosphonylmethoxyethyl)guanine) is an acyclic nucleotide analog being evaluated for its anti-proliferative activity. We examined the inhibitory effects of PMEG diphosphate (PMEGpp) toward DNA polymerases (pol) delta and epsilon and found it to be a competitive inhibitor of both these enzymes. The apparent Ki values for PMEGpp were 3-4 times lower than the Km values for dGTP. The analog was shown to function as a substrate and to be incorporated into DNA by both enzymes. Examination of the ability of pol delta and pol epsilon to repair the incorporated PMEG revealed that pol epsilon could elongate PMEG-terminated primers in both matched and mismatched positions with an efficiency equal to 27 and 85% that observed for dGMP-terminated control template-primers. Because PMEG acts as an absolute DNA chain terminator, the elongation of PMEG-terminated primers is possible only by cooperation of the 3'-5'-exonuclease and DNA polymerase activities of the enzyme. In contrast to pol epsilon, pol delta exhibited negligible activity on these template-primers, indicating that pol epsilon, but not pol delta, can repair the incorporated analog.
Kramata
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Gilead Sciences, Foster City, California 94404, USA. Pavel_Kramata@gilead.com
Downey
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Paborsky
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LR
D000818:Animals; D002417:Cattle; D004257:DNA Polymerase II / Q000378:metabolism*; D004258:DNA Polymerase III / Q000378:metabolism*; D017931:DNA Primers / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D008956:Models, Chemical; D009841:Oligonucleotides / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism*; D013698:Templates, Genetic
D017931:DNA Primers; D009841:Oligonucleotides; D009943:Organophosphorus Compounds; C063380:9-((2-phosphonylmethoxy)ethyl)guanine; D006147:Guanine; D004257:DNA Polymerase II; D004258:DNA Polymerase III