BASE ANALOG AND NEIGHBORING BASE EFFECTS ON SUBSTRATE-SPECIFICITY OF RECOMBINANT HUMAN G-T MISMATCH-SPECIFIC THYMINE DNA-GLYCOSYLASE

We studied the substrate specificity of the human G:T mismatch-specifi c thymine glycosylase that initiates the repair of G:T and G:U base mi smatches to G:C base pairs. Such mismatches arise when 5-methylcytosin e or cytosine deaminate spontaneously (and hydrolytically) in DNA. Sub strates were 45-bp DNA heteroduplexes that bore single G:T, m6G:T, 2,6 -diaminopurine:T, 2-amino-6-(methylamino)purine:T, 2-aminapurine:T, an d G:m4T mispairs, The bases 5' to the poorly matched G were altered in selected G:T substrates to yield mispairs in four different contexts, ApG, CpG, CpG and TpG. The recombinant thymine glycosylase was incuba ted with the 45-bp DNA substrates, each labeled at the 5'-terminus of the strand containing the mismatched T. The DNAs were then treated wit h 0.1 N NaOH to catalyze phosphodiester bond breakage at the newly-gen erated AP sires, and the products were analyzed on DNA sequencing gels . As indicated by the amounts of the 20-nt incision product, the remov al of the thymine base by the enzyme increased linearly between 0 and 40 min at which time the generation of product from all substrates cea sed, probably because of enzyme inactivation. The rate of incision was greatest (0.7 fmol/min) with DNA containing The G:T mispair followed by the DNA containing the m6G:T mispair (0.38 fmol/min) and the DNA wi th the 2-amino-6-(methylamino)purine:T mispair (0.15 fmol/min); the ex tent of reaction was 90%, 40%, and 20% respectively. By contrast to pr evious findings with cell-free extracts, DNA substrates containing 2,6 -diaminopurine:T, 2-aminopurine:T, and G:m4T mispairs were not incised (<2%). The amount of incision of the 45-bp DNA substrates containing G:T mispairs in the CpG context was 3-12-fold greater than in the TpG, GpG, and ApG contexts.