Elucidating the role of mismatch repair in class switch recombination and chromatin assembly

Abstract

The successful survival of a species depends on faithful passage of genetic material from one cell to its daughters. Replicative polymerases possess low error rates, but, in spite of this, 1/107 nucleotides escape their proof reading activity. These biosynthetic errors have to be corrected by postreplicative mismatch repair (MMR), which improves replication fidelity by two to three orders of magnitude. The importance of MMR is beyond doubt – its malfunction brings about a mutator phenotype, which was shown to be the underlying cause of Lynch Syndrome, a predisposition to early-onset cancer of the colon, endometrium, ovary and other organs. We hypothesized that MMR might interfere with chromatin packaging, as rapid assembly of nucleosomes behind the replication fork would most likely hinder the mismatch recognition factor MutSa from binding mismatches and initiating their repair. To study the interplay of MMR and chromatin assembly, we set up a biochemical system using human cell extracts that were proficient for both chromatin assembly and MMR. As anticipated, mismatch-containing plasmids carrying preassembled nucleosomes were poor substrates for MMR. In contrast, ongoing MMR interfered with nucleosome deposition. CAF-1, the mediator of chromatin assembly, and the MSH6 subunit of the mismatch recognition factor MutSa both interact with proliferating cell nuclear antigen (PCNA), the processivity factor of replicative DNA polymerases. We therefore postulated that PCNA might govern the balance between MMR and chromatin assembly. We found, however, that this regulation might be more complex than foreseen, as MutSa and CAF-1 interact not only with PCNA, but also with each other. Recent literature implicated MMR proteins, in addition to the repair of biosynthetic errors, also in DNA damage response, triplet repeat stability, mitotic and meiotic recombination, the repair of interstrand cross-links and antibody diversification. Antibody diversification consists of three processes affecting immunoglobulin (Ig) loci: V(D)J recombination, somatic hypermutation (SHM) and class switch recombination (CSR). CSR endows antibodies with different effector functions. It is initiated by activation-induced cytidine deaminase (AID), which converts cytosines to uracils and thus gives rise to U/G mispairs. Surprisingly, metabolism of U/Gs in activated B-cells is inefficient and gives rise to DNA double strand breaks (DSBs), one of the key prerequisites for CSR. Genetic data implicate two DNA repair pathways in the processing of these mismatches: base excision repair (BER) and mismatch repair (MMR). In order to dissect the molecular mechanism of the roles of these repair pathways in the processing of U/G mispairs, we generated substrates containing uracils at defined positions and incubated them with extracts of human cells. We found that the induction of DSBs was dependent on the BER enzyme uracil N-glycosylase (UNG) and on the MMR factor MutSa, whereas MutLa was only found to participate in a subset of events and its contribution depended on its endonucleolytic activity. Given that interference of BER and MMR is not restricted to B-cells, it may pose a general threat to genomic integrity.