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MutL stimulates the DNA duplex unwinding activity of UvrD in methyl-directed DNA mismatch repair (MMR) via their physical interactions. However, the molecular functions of MutL associated with the DNA binding and UvrD helicase have been partially understood. We present the kinetic characteristics of the single-stranded DNA (ssDNA) binding activity of MutL in the absence or the presence of UvrD helicases using the single-molecule techniques. The lengthening of the ssDNA due to the ssDNA binding of MutL allows us to observe association and dissociation of MutL from the ssDNA in real-time. In this study, we demonstrate that the nonspecific ssDNA binding of MutL can be involved in subsequent loading of UvrD helicases onto the ssDNA in a manner independent of ATP hydrolysis of MutL.

The crystal structure of the DNA heptamer d(GCGCGCG) has been solved at 1.65 A resolution by the molecular replacement method and refined to an R-value of 0.184 for 3598 reflections. The heptamer forms a Z-DNA d(CGCGCG)2 with 5'-overhang G residues instead of an A-DNA d(GCGCGC)2 with 3'-overhang G residues. The overhang G residues from parallel strands of two adjacent duplexes form a trans reverse Hoogsteen G x G basepair that stacks on the six Z-DNA basepairs to produce a pseudocontinuous helix. The reverse Hoogsteen G x G basepair is unusual in that the displacement of one G base relative to the other allows them to participate in a bifurcated (G1)N2 . . . N7(G8) and an enhanced (G8)C8-H . . . O6(G1) hydrogen bond, in addition to the two usual hydrogen bonds. The 5'-overhang G residues are anti and C2'-endo while the 3'-terminal G residues are syn and C2'-endo. The conformations of both G residues are different from the syn/C3'-endo for the guanosine in a standard Z-DNA. The two cobalt hexammine ions bind to the phosphate groups in both GpC and CpG steps in Z(I) and Z(II) conformations. The water structure motif is similar to the other Z-DNA structures.

The crystal structure of the alternating 5'-purine start decamer d(GCGCGCGCGC) was found to be in the left-handed Z-DNA conformation. Inasmuch as the A.T base pair is known to resist Z-DNA formation, we substituted A.T base pairs in the dyad-related positions of the decamer duplex. The alternating self-complementary decamer d(GCACGCGTGC) crystallizes in a different hexagonal space group, P6(1)22, with very different unit cell dimensions a = b = 38.97 and c = 77.34 A compared with the all-G.C alternating decamer. The A.T-containing decamer has one strand in the asymmetric unit, and because it is isomorphous to some other A-DNA decamers it was considered also to be right-handed. The structure was refined, starting with the atomic coordinates of the A-DNA decamer d(GCGGGCCCGC), by use of 2491 unique reflections out to 1.9-A resolution. The refinement converged to an R value of 18.6% for a total of 202 nucleotide atoms and 32 water molecules. This research further demonstrates that A.T base pairs not only resist the formation of Z-DNA but can also assist the formation of A-DNA by switching the helix handedness when the oligomer starts with a 5'-purine; also, the length of the inner Z-DNA stretch (d(CG)n) is reduced from an octamer to a tetramer. It may be noted that these oligonucleotide properties are in crystals and not necessarily in solutions.

It has been studied that DNA repair proteins search a target via a 1-dimensional diffusion along naked DNA. Due to the absence of the target on DNA this single-molecule tracking approach lacked of understanding the catalytic function of the repair proteins and moreover the mechanism of the downstream transactions for the repair of an error on the DNA. We present the catalytic processes of MutS, mismatch repair initiation protein, on a mismatched DNA that bears an unpaired nucleotide. Our single-molecule analysis reveals that MutS undergoes two distinct conformational changes during DNA mismatch repair (MMR). MutS forms a transient clamp that scans duplex DNA for the unpaired nucleotide and a different sliding clamp that is induced by ATP binding to the mismatch-bound MutS with unusual stability on DNA. These observations suggest a mechanism of how MMR machinery can be recruited for the strand incision, which is highly controversial.