hMutα forms an ATP-dependent complex with hMutLα and hMutLβ on DNA

The DNA binding properties of hMutSalpha and hMutLalpha and complex formation of hMutSalpha with hMutLalpha and hMutLbeta were investigated using binding experiments on magnetic bead-coupled DNA substrates with nuclear extracts as well as purified proteins. hMutSalpha binding to homoduplex DNA was disrupted by lower NaCl concentrations than hMutSalpha binding to a mismatch. ATP markedly reduced the salt resistance of hMutSalpha binding but hMutSalpha still retained affinity for heteroduplexes. hMutSalpha formed a complex with hMutLalpha and hMutLbeta on DNA in the presence of ATP. This complex only formed on 81mer and not 32mer DNA substrates. Complex formation was enhanced by a mismatch in the DNA substrate, and hMutLalpha and hMutLbeta were shown to enter the complex at different ATP concentrations. Purified hMutLalpha showed an intrinsic affinity for DNA, with a preference for single-stranded over double-stranded DNA.