The human MutS homologues (MSH), hMSH2 and hMSH6, forms a heterodimer (hMSH2-hMSH6) that plays a central role in mismatch repair (MMR). hMSH2-hMSH6 is required for the recognition of mismatched nucleotides and insertion/deletion loops (IDLs) generated by misincorporation during DNA replication. Mutations in either of the hMSH2 or hMSH6 genes result in elevated spontaneous mutation rate and susceptibility to the common cancer predisposition syndrome, Lynch Syndrome or hereditary non-polyposis colorectal cancer (LS/HNPCC).
Mismatches that are recognized by hMSH2-hMSH6 arise in vivo within chromosomes that are a complex mixture of DNA and protein (chromatin). A fundamental unit of chromatin is the nucleosome which consists of ~147 bp of DNA wrapped twice around a histone octamer containing two H2A-H2B dimers and an H3-H4 tetramer. The biophysical/biochemical effect of chromatin on MMR is unknown. Moreover, little is known about the effect of more than a 100 post-translational modifications (PTMs) that may decorate the human histones during MMR processes. This dissertation discusses chromatin and MMR. Chapter 1 serves as an introduction to MMR and chromatin. Chapter 2 provides the thesis rationale.
Chapter 3 involves analysis of a mismatched DNA substrate containing a single well-defined nucleosome. We demonstrate that hMSH2-hMSH6 can catalyze the disassembly of a nucleosome adjacent to a mismatch. In addition, we have constructed nucleosomes containing acetylations of the histone H3 dyad residues K115 and K122 by a semi-synthetic intein-based strategy. We find that hMSH2-hMSH6 nucleosome disassembly is considerably enhanced when nucleosomes contain H3(K115, K122) acetylation modifications. Moreover, lysine to a glutamine substitution mutation of histone H3(K56), used to mimic the lysine acetylation, also enhances nucleosome disassembly. Disassembly of the nucleosome requires ATP binding by hMSH2-hMSH6. In addition, nucleosome disassembly is blocked by LacI/LacO placed between the mismatch and the nucleosome arguing in favor of a “cis” or “moving” mechanism.
Chapter 4 is devoted to analyzing single acetylation and mimics of the acetylation of histone H3(K115) and/or H3(K122). We observe that hMSH2-hMSH6 nucleosome disassembly is enhanced with acetylation and mimicked acetylation modifications of H3(K115) and/or H3(K122). Moreover, hMSH2-hMSH6 nucleosome disassembly is dependent on the nucleosome positioning sequence (NPS). hMSH2-hMSH6 nucleosome disassembly is considerably enhanced with the physiological relevant Xenopus 5S rDNA NPS. Disassembly of the nucleosome by hMSH2-hMSH6 is masked by the high affinity nonphysiological 601 and pMP2 NPS’s. Replacement of the DNA sequence at the nucleosomal-dyad axis in the Xenopus 5S rDNA NPS with the 601 NPS reduces nucleosome disassembly by hMSH2-hMSH6 ~2-fold. We also find that hMSH2-hMSH6 can recognize and bind to a mismatch within the nucleosome. hMSH2-hMSH6 binding affinity and nucleosome disassembly is increased when the mismatch is located at the entry-exit region of the nucleosome compared to a mismatch located at the LRS (loss of rDNA silencing) or the nucleosomal-dyad axis region. Furthermore, the ability of hMSH2-hMSH6 to disassemble nucleosomes containing mismatches is enhanced when nucleosomes contain H3(K115, K122) acetylation modifications. These results highlight that nucleosome disassembly by hMSH2-hMSH6 is dependent on NPS and histone octamer modifications. Moreover, acetylation/mimic modifications enhance hMSH2-hMSH6 nucleosome disassembly.
Chapter 5 focuses on histone H3(K56) acetylation which occurs during DNA replication and repair and is located in the entry-exit region of the nucleosome. We find that a single nucleosome containing a H3(K56) acetylation with an adjacent mismatch is disassembled by hMSH2-hMSH6.
Chapter 6 is centered on histone H3(T118) phosphorylation, which is located at the nucleosomal-dyad axis region of the histone-DNA interface, directly adjacent to the DNA backbone. We analyzed a single nucleosome reconstituted on the high affinity pMP2 artificial sequence containing an adjacent mismatch and observed enhanced hMSH2-hMSH6 nucleosome disassembly compared to unmodified nucleosomes reconstituted on the pMP2 sequence. The results detailed in this thesis improve our understanding of MMR in the context of chromatin. Together, these results suggest a novel passive mechanism for nucleosome disassembly by hMSH2-hMSH6.