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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.

Original publication

DOI

10.1093/nar/28.13.2535

Type

Journal article

Journal

Nucleic Acids Res

Publication Date

01/07/2000

Volume

28

Pages

2535 - 2540

Keywords

2-Aminopurine, Base Pair Mismatch, Base Pairing, Base Sequence, DNA, Deoxyuridine, Endodeoxyribonucleases, Escherichia coli, Guanine, Kinetics, Purine Nucleosides, Ribonucleosides, Substrate Specificity, Thymine, Toluene