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-specific thymine glycosylase that initiates the repair of G:T and G:U base mismatches to G:C base pairs. Such mismatches arise when 5-methylcytosine or cytosine deaminate spontaneously (and hydrolytically) in DNA. Substrates were 45-bp DNA heteroduplexes that bore single G:T, m6G:T, 2,6-diaminopurine:T, 2-amino-6-(methylamino)-purine:T, 2-aminopurine:T, and 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, GpG, and TpG. The recombinant thymine glycosylase was incubated with the 45-bp DNA substrates, each labeled at the 5'-terminus of the strand containing the mismatched T. The DNAs were then treated with 0.1 N NaOH to catalyze phosphodiester bond breakage at the newly-generated AP sites, and the products were analyzed on DNA sequencing gels. As indicated by the amounts of the 20-nt incision product, the removal of the thymine base by the enzyme increased linearly between 0 and 40 min at which time the generation of product from all substrates ceased, 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 with the 2-amino-6-(methylamino)purine:T mispair (0.15 fmol/ min); the extent of reaction was 90%, 40%, and 20% respectively. By contrast to previous 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.
Sibghat-Ullah
Molecular Oncology Program, Cross Cancer Institute, Edmonton, Alberta, Canada.
Gallinari
P
P
Xu
Y Z
YZ
Goodman
M F
MF
Bloom
L B
LB
Jiricny
J
J
Day
R S
RS
D044503:5-Methylcytosine; D001482:Base Composition; D003596:Cytosine / Q000031:analogs & derivatives / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer)