DNA binding and cleavage selectivity of the Escherichia coli DNA G : T-mismatch endonuclease (vsr protein)

The Escherichia coli vsr endonuclease recognises T:G base-pair mismatches i n double-stranded DNA and initiates a repair pathway by hydrolysing the pho sphate group 5 ' to the incorrectly paired T. The gene encoding the vsr end onuclease is next to the gene specifying the E. coil dent DNA-methyltransfe rase; an enzyme that adds CH3 groups to the first dC within its target sequ ence CC[A/T]GG, giving (CC)-C-5Me[A/T]GG. Deamination of the d(5Me)C result s in CT[A/T]GG in which the first T is mis-paired with de and it is believe d that the endonuclease preferentially recognises T:G mismatches within the dcm recognition site. Here, the preference of the vsr endonuclease for bas es surrounding the T:G mismatch has been evaluated. Determination of specif icity constant (k(st)/K-D; k(st) = rate constant for single turnover, K-D = equilibrium dissociation constant) confirms vsr's preference for a T:G mis match within a dcm sequence i.e. C (T) under bar [A/T]GG (the underlined T being mis-paired with de) is the best substrate. However, the enzyme is cap able of binding and hydrolysing sequences that differ from the dcm target s ite by a single base-pair (dcm star sites). Individual alteration of any of the four bases surrounding the mismatched T gives a substrate, albeit with reduced binding affinity and slowed turnover rates. The vsr endonuclease h as a much lower selectivity for the dcm sequence than type II restriction e ndonucleases have for their target sites. The results are discussed in the light of the known crystal structure of the vsr protein and its possible ph ysiological role. (C) 2001 Academic Press.