Figure 5.

Chemical crosslinking SC-MutH^A223C to SC-MutL^480C abolishes MutS requirement for MutH activation. (A) SDS–PAGE analysis of HPLC-purified complex SC-MutH^A223C with SC-MutL^480C crosslinked with the cleavable reagent MTS-11-O3-MTS (for details see Materials and Methods) before and after treatment with 5 mM DTT. The amount of uncrosslinked SC-MutH^A223C co-purifying with the complex was judged to be <5% of the crosslinked MutH. (B) MutH DNA cleavage promoted by MutL and MutS was assayed as described in Materials and Methods using a 484 bp heteroduplex DNA (25 nM). The complex of SC-MutH^A223C/SC-MutL^480C (1 µM) crosslinked (−DTT, open symbols) or uncrosslinked (+DTT, closed symbols) was assayed in the absence (triangles) or presence (squares) of 1 µM MutS. Note that only in the crosslinked complex MutH is able to cleave DNA even in the absence of MutS.