Structural and mechanistic studies Of DNA mismatch repair

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Mismatch repair (MMR) in E. coli is initiated by three proteins, MutS, MutL and MutH to specifically target newly synthesized daughter strand. MutS is an ATPase and recognizes a mismatched base-pair as well as an insertion or deletion of 1-4 nucleotides in one strand. MutH is a latent endonuclease that is both sequence- and methylation-specific; when activated by MutS upon detection of a mismatch, it cleaves 5 to the unmethylated d(GATC) sequence in a hemimethylated duplex. MutL is also an ATPase and mediates the communication between MutS and MutH, which do not directly interact. Once a nick is introduced to the daughter strand by MutH, UvrD helicase, single-strand binding protein and DNA exonuclease are recruited by MutS and MutL to remove nucleotides from the nick to beyond the mismatch. Homologues of MutS and MutL are found in all eukaryotes, and malfunction of either human MutS or MutL homolog is directly implicated in the susceptibility to hereditary non-polyposis colorectal cancer (HNPCC) and other sporadic cancers. Our previous studies led to the determination of crystal structures of MutS, MutS-mismatch DNA and MutS-mismatch-ADP complexes, the N- and C-terminal domain of MutL, and finally MutH and MutH-DNA complexes. We also characterized the role of the MutS and MutL ATPases and the cleavage specificity of MutH. In this fiscal year, we have succeeded in determining a series of crystal structures of UvrD helicase-DNA complexes, which represent consecutive physical steps of UvrD unwinding a duplex DNA in an ATP hydrolysis cycle. In addition, we have carried out mutagenesis studies to dissect two alternative mechanisms of DNA unwinding by UvrD. Our manuscript UvrD helicase unwinds DNA one base pair at a time by a two-part power stroke was published in Cell in December 2006. Since 2016, we attempting to (1) obtain large protein-DNA assemblies, e.g. MutL-DNA, MutL-UvrD-DNA, MutS-MutL-DNA complexes, for structural characterization, (2) figure out how MMR proteins MutSbeta and MutL cooperate to drive trinucleotide repeat expansions. MutS proteins bind to and bend DNA at DNA mismatches located within a normal double helix 16,21,44,45 (Fig. 1a); it has been suggested that (1) MutS binds to the hairpin end of an extruded hairpin of CNG repeats while anchoring on the surrounding DNA duplex 46, and (2) the binding of several MutS molecules to a cluster of such extruded hairpins prevents proper mismatch repair (Fig. 1c) 47. Since it is energetically less favorable to form multiple rather than a single hairpin extrusion from perfectly base-paired CNG repeats and no one has visualized the association of MutS with TNR repeats, we examined the binding of MutS to, and the structures of, all CNG (N=A, C, G or T) and GAA repeats bound by human MutS without any surrounding DNA double helix (Fig. 1d) Contrary to all previous predictions, we found that MutS prefers binding to the stem of an extruded (CNG) or GAA hairpin with sub-nanomolar dissociation constant, rather than to the hairpin end or hairpin-duplex junction. Structural analyses reveal that in the presence of MutS, CNG repeats with N:N mismatches adopt a B form-like pseudo-duplex, with one or two CNG repeats slipped out, forming uneven bubbles that partly mimic insertion-deletion loops of mismatched DNA (Fig. 1e-f) 48. When the length of extruded hairpin exceeds the threshold of 40 repeats, each hairpin can be bound by three or more MutS molecules, which are resistant to ATP-dependent dissociation (Fig. 3a-c). Therefore, we hypothesize that such MutS-CNG complexes recruit MutL endonuclease to nick DNA and initiate the repeat expansion process. Our unexpected finding of MutS binding to CNG pseudo-duplexes with every third base pair being an N:N mismatch provides a key distinction between the essential function of MutS in replication-associated mismatch repair and its pathological role in replication-independent repeat expansions. This discovery immediately suggests a simple screen for inhibitors that can prevent the pathologic binding of MutS to CNG repeats without affecting its usual mismatch recognition. As a proof of principle, we demonstrated that Actinomycin D (ActD), which prefers to intercalate at T:T mismatches and stabilize distorted and non-B DNA structure 51,52, inhibits MutS binding to CNG repeats. At 300 nM, ActD completely abolishes MutS binding to CTG, CCG and CAG repeats, but does not interfere with the recognition of IDLs by MutS 48. Although the toxicity of ActD and its inability to cross the blood-brain barrier prevent its use in treating patients with repeat expansions, the straightforward inhibitor screening approach offers a promising method to discover effective and specific inhibitors for TNR expansions. In the current fiscal year, in collaboration with Dr. Zheng's group at NCATS, we have completed the first screen of the FDA-approved small molecular library. We are moving onto virtual screening of a much larger library based on the initial screening results. Hopefully we will have hits to pursue in test-tube and cell in the coming year. References Li, J., Wang, H and Yang, W. (2024) Tandem MutSβ binding to long extruded DNA trinucleotide repeats underpins pathogenic expansions. BioRxiv, doi: 10.1101/2023.12.12.571350. Ortega, J., Lee, G.S., Gu, L., Yang, W. & Li, G.M. (2021) Mispair-bound human MutS-MutL complex triggers DNA incisions and activates mismatch repair. Cell Research, 31, 542-553. Gu, H. Z., Yang, W. & Seeman, N. C. (2010). DNA scissors device used to measure MutS binding to DNA mis-pairs, JACS, 132, p4252-4357. Gupta, S., Gellert, M. & Yang , W. (2011) Mechanism of mismatch recognition revealed by human MutSbeta bound to unpaired DNA loops. NSMB. 19, 72-78. Yang, W. (2010) Lessons learnt from UvrD helicase: mechanism for directional movement. Ann. Rev. Biophys., 39, 367-385.