Recognition of GT mismatches by Vsr mismatch endonuclease.
The Vsr mismatch endonuclease recognises the sequence CTWGG (W = A or T) in which the underlined thymine is paired with guanine and nicks the DNA backbone on the 5'-side of the mispaired thymine. By using base analogues of G and T we have explored the functional groups on the mismatch pair which are recognised by the enzyme. Removal of the thymine 5-methyl group causes a 60% reduction in activity, while removing the 2-amino group of guanine reduces cleavage by 90%. Placing 2-amino-purine or nebularine opposite T generates mis-matches which are cut at a much lower rate (0.1%). When either base is removed, generating a pseudoabasic site (1', 2'-dideoxyribose), the enzyme still produces site-specific cleavage, but at only 1% of the original rate. Although TT and CT mismatches at this position are cleaved at a low rate (approximately 1%), mismatches with other bases (such as GA and AC) and Watson-Crick base pairs are not cleaved by the enzyme. There is also no cleavage when the mismatched T is replaced with difluorotoluene.
Fox
K R
KR
Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, UK. krf1@soton.ac.uk
Allinson
S L
SL
Sahagun-Krause
H
H
Brown
T
T
D015075:2-Aminopurine / Q000378:metabolism; D020137:Base Pair Mismatch / Q000235:genetics*; D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003857:Deoxyuridine / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism*; D007700:Kinetics; D011684:Purine Nucleosides / Q000378:metabolism; D012263:Ribonucleosides / Q000378:metabolism; D013379:Substrate Specificity; D013941:Thymine / Q000031:analogs & derivatives / Q000378:metabolism*; D014050:Toluene / Q000031:analogs & derivatives* / Q000378:metabolism
D011684:Purine Nucleosides; D012263:Ribonucleosides; D014050:Toluene; D015075:2-Aminopurine; C110527:difluorotoluene; C024829:8-hydroxyguanine; D006147:Guanine; D004247:DNA; C005248:nebularine; D004706:Endodeoxyribonucleases; C071421:vsr endonuclease; D013941:Thymine; D003857:Deoxyuridine