New noncleavable analogs of 8-oxoguanine-DNA glycosylase substratesстатья
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Дата последнего поиска статьи во внешних источниках: 18 июля 2013 г.
Аннотация:8-Oxoguanine-DNA glycosylases play a key role in repairing oxidatively damaged DNA. Excision repair enzymes Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg protein) and human 8-oxoguanine-DNA glycosylase (hOGG1) catalyze excision of 7,8-dihydro-8-oxoguanine (oxoG) from DNA and subsequent cleavage of the sugar–phosphate backbone. Contacts between DNA phosphate groups and amino acid residues of the active centers of the enzymes are of importance for specific binding and catalysis. To construct noncleavable analogs of Fpg protein and hOGG1 substrates, modifications of phosphate groups bound to a damaged nucleotide were tested for their effect on the substrate properties of modified DNA duplexes. New oxoG-containing analogs of Fpg protein and hOGG1 substrates were synthetic DNA duplexes that contained a pyrophosphate or a substituted pyrophosphate group bound with the 5′- or 3′-OH of 8-oxoguanosine. The duplexes proved to be recognized and specifically bound by Fpg protein and hOGG1. Analysis of the mechanism of their interaction with Fpg protein and hOGG1 showed that modification of the internucleotide phosphate bound with 3′-OH of 8-oxoguanosine prevents oxoG excision from DNA. Yet both enzymes efficiently cleaved the DNA duplexes when the modified phosphate was bound with the 5′-OH of 8-oxoguanosine. DNA duplexes with a pyrophosphate or substituted pyrophosphate group at 3′-OH of 8-oxoguanosine are noncleavable analogs of 8-oxoguanine-DNA glycosylase substrates and can be used to study the structures of catalytically active forms of Fpg protein and hOGG1 and their prokaryotic or eukaryotic homologs in complex with oxoG-containing DNA.