Nucleic Acids Research, 2000, Vol. 28, No. 13 2535-2540
© 2000 Oxford University Press
Recognition of GT mismatches by Vsr mismatch endonuclease
1Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK and 2Department of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, UK
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-aminopurine or nebularine opposite T generates mismatches 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 (~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.
* To whom correspondence should be addressed. Tel: +44 2380 594374; Fax: +44 2380 594459; Email: krf1@soton.ac.uk The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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