Mismatch repair in human nuclear extracts - Effects of internal DNA-hairpin structures between mismatches and excision-initiation nicks on mismatch correction and mismatch-provoked excision

DNA mismatch repair (MMR) couples recognition of base mispairs by MSH2.MSH6 heterodimers to initiation, hundreds of nucleotides away, of nascent strand 3'-5' or 5'-3' excision through the mispair. Mismatch-recognition complexes have been hypothesized to move along DNA to excision-initiation signals, in eukaryotes, perhaps ends of nascent DNA, or to remain at mismatches and search through space for initiation signals. Subsequent MMR excision, whether simple processive digestion of the targeted strand or tracking of an excision complex, remains poorly understood. In human cell-free extracts, we analyzed correction of a mismatch in a 2.2-kilobase pair (kbp) circular plasmid containing a pre-existing excision-initiation nick for initiation, and measured MMR excision (in the absence of exogenous dNTPs) at specific locations. Excision specificities were similar to100:1 for nicked versus continuous strands, 80:1 for mismatched versus homoduplex DNA, and 30:1 for shorter (0.3-kbp) versus longer (1.9-kbp) nick-mispair paths. To test models for recognition-excision coupling and excision progress, we inserted potential blockades, 20-bp hairpins, into nick-mispair paths, using a novel technique to first generate gapped plasmid. Continuous strand longer-path hairpins did not affect mismatch correction, but shorter-path hairpins reduced correction 4-fold, and both together eliminated it. Shorter-path hairpins had little effect on initiation of (3'-5') excision, measured 30-60 nucleotides 5' to the nick, but blocked subsequent progress of excision to the mismatch; longer-path hairpins blocked the (lower level) 5'-3' excision to the mismatch. Thus, (a) MMR excision protein(s) cannot move past DNA hairpins. Hairpins at both ends of substrate-derived 0.5-kbp DNA fragments did not prevent ATP-induced dissociation of mismatch-bound human MSH2.MSH6, so recognition complexes at mismatches might provoke excision at nicks beyond hairpins, or loosely sliding MSH2.MSH6 dimers might move to the nicks.