ROLES FOR THE INTRINSICALLY DISORDERED LINKER ARMS OF MLH PROTEINS IN DNA MISMATCH REPAIR
Furman, Christopher Michael
Eukaryotic mismatch repair (MMR) is initiated when a misincorporation event occurring during DNA replication is recognized by MutS homolog (MSH) heterodimeric proteins. MSH complexes then recruit MutL homolog (MLH) heterodimeric complexes. Once recruited to the MSH-mismatch site, MLH complexes undergo ATP-dependent conformational changes that results in the nicking of the newly replicated strand. This nicking step provides entry for downstream repair factors that subsequently excise and repair the error. MLH proteins contain long and flexible intrinsically disorder regions (IDRs) that connect structured N- and C- terminal domains. I characterized how IDRs regulate the functions of the yeast Mlh1-Pms1 mismatch repair (MMR) complex. Shortening or scrambling the IDRs in both subunits ablated MMR in vivo. Mlh1-Pms1 complexes with shorter IDRs that disrupt MMR retained wild-type DNA binding affinity but were impaired for diffusion on both naked and nucleosome-coated DNA. Moreover, the IDRs also regulated the ATPase and nuclease activities encoded in the structured N- and C-terminal domains of the complex. This combination of phenotypes underlies the catastrophic MMR defect seen with the mutant Mlh1-Pms1 in vivo. More broadly, this work highlighted an unanticipated multi-functional role for IDRs in regulating both facilitated diffusion on chromatin and nucleolytic processing of a DNA substrate. These studies encouraged me to perform a set of experiments where I inserted FRB and FKBP dimerization domains at various positions within the Mlh1 and Pms1 IDRs that did not disrupt MMR. I then induced rapamycin-dependent FRB-FKBP interactions both in vivo and in vitro. Through this approach, I created an mlh1 allele that can be reversibly disrupted for MMR upon rapamycin treatment, providing a new tool to disrupt MMR on demand. I then showed that restraining the MLH linkers disrupted the coordinated movement of the N-terminal MLH ATP binding domains and caused defects in MMR. In contrast, restraints predicted to maintain free movement of the N-terminal ATP binding domains had weak effects on MMR. Restriction of coordinated movements disrupted DNA and PCNA dependent regulation of Mlh1-Pms1 ATPase activity and Mlh1-Pms1 DNA binding affinity. Together, this work provides support for MLH linker domains mediating distinct conformational steps in DNA MMR and is consistent with a two-step clamp model for MLH proteins acting in eukaryotic MMR. Additionally, I performed a separate set of studies aimed at understanding how Mlh1-Mlh3 acts in its major role to resolve double Holliday junctions into crossovers that facilitate the Meiosis I division. Through a phylogenetic approach, I identified residues in baker’s yeast Mlh3 critical for its meiotic functions. Sites in Mlh3 were changed to the Pms1 equivalent in conserved positions located outside of motifs found in all MLH family members. I also made changes in sites that are conserved in one family but missing from the other, and constructed Mlh3/Pms1 chimeras. The resulting strains showed phenotypes similar to mlh3 hypomorph or null alleles, and in some cases showed phenotypes stronger than the mlh3 null, providing evidence that all three domains in the MLH protein family are critical for conferring pathway specificity. Importantly, mlh3 ATP binding and endonuclease domain alleles improved MMR functions in pms1Δ strains without disrupting meiotic functions, indicating an expansion of Mlh3 functions, and suggesting that MLH proteins have both mismatch repair and crossover functions. This strategy provides an approach to understand how paralogs have evolved to support distinct cellular processes.
IDRs; Mismatch repair; MutL Homologs
Smolka, Marcus B.; Peters, Joe E.
Biochemistry, Molecular and Cell Biology
Ph. D., Biochemistry, Molecular and Cell Biology
Doctor of Philosophy
dissertation or thesis