Biochemical characterization of a cancer-associated E109K missense variant of human exonuclease 1

Mutations in the mismatch repair (MMR) genes MSH2, MSH6, MLH1 and PMS2 are associated with Lynch Syndrome (LS), a familial predisposition to early-onset cancer of the colon and other organs. Because not all LS families carry mutations in these four genes, the search for cancer-associated mutations was extended to genes encoding other members of the mismatch repairosome. This effort identified mutations in EXO1, which encodes the sole exonuclease implicated in MMR. One of these mutations, E109K, was reported to abrogate the catalytic activity of the enzyme, yet, in the crystal structure of the EXO1/DNA complex, this glutamate is far away from both DNA and the catalytic site of the enzyme. In an attempt to elucidate the reason underlying the putative loss of function of this variant, we expressed it in Escherichia coli, and tested its activity in a series of biochemical assays. We now report that, contrary to earlier reports, and unlike the catalytic site mutant D173A, the EXO1 E109K variant resembled the wild-type (wt) enzyme on all tested substrates. In the light of our findings, we attempt here to reinterpret the results of the phenotypic characterization of a knock-in mouse carrying the E109K mutation and cells derived from i

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

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

Non-canonical uracil processing in DNA gives rise to double-strand breaks and deletions: relevance to class switch recombination

During class switch recombination (CSR), antigen-stimulated B-cells rearrange their immunoglobulin constant heavy chain (CH) loci to generate antibodies with different effector functions. CSR is initiated by activation-induced deaminase (AID), which converts cytosines in switch (S) regions, repetitive sequences flanking the CHloci, to uracils. Although U/G mispairs arising in this way are generally efficiently repaired to C/Gs by uracil DNA glycosylase (UNG)-initiated base excision repair (BER), uracil processing in S-regions of activated B-cells occasionally gives rise to double strand breaks (DSBs), which trigger CSR. Surprisingly, genetic experiments revealed that CSR is dependent not only on AID and UNG, but also on mismatch repair (MMR). To elucidate the role of MMR in CSR, we studied the processing of uracil-containing DNA substrates in extracts of MMR-proficient and -deficient human cells, as well as in a system reconstituted from recombinant BER and MMR proteins. Here, we show that the interplay of these repair systems gives rise to DSBsin vitroand to genomic deletions and mutationsin vivo, particularly in an S-region sequence. Our findings further suggest that MMR affects pathway choice in DSB repair. Given its amenability to manipulation, our system represents a powerful tool for the molecular dissection of CSR

Noncanonical Mismatch Repair as a Source of Genomic Instability in Human Cells

Mismatch repair (MMR) is a key antimutagenic process that increases the fidelity of DNA replication and recombination. Yet genetic experiments showed that MMR is required for antibody maturation, a process during which the immunoglobulin loci of antigen-stimulated B cells undergo extensive mutagenesis and rearrangements. In an attempt to elucidate the mechanism underlying the latter events, we set out to search for conditions that compromise MMR fidelity. Here, we describe noncanonical MMR (ncMMR), a process in which the MMR pathway is activated by various DNA lesions rather than by mispairs. ncMMR is largely independent of DNA replication, lacks strand directionality, triggers PCNA monoubiquitylation, and promotes recruitment of the error-prone polymerase-η to chromatin. Importantly, ncMMR is not limited to B cells but occurs also in other cell types. Moreover, it contributes to mutagenesis induced by alkylating agents. Activation of ncMMR may therefore play a role in genomic instability and cancer