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.     

Preferential accessibility to specific genomic loci for the repair of double-strand breaks in human cells

The dynamic organization of the human genome in the nucleus is gaining recognition as a determining factor in its functional regulation. In order to be expressed, replicated or repaired, a genomic locus has to be present at the right place at the right time. In the present study, we have investigated the choice of a double-strand break (DSB) repair partner for a given genomic loci in an ATM-deficient human fibroblast cell line. We found that partner choice is restricted such that a given genomic locus preferentially uses certain sites in the genome to repair itself. These preferential sites can be in the vicinity of the damage site or megabases away or on other chromosomes entirely, while potential sites closer to the break along the length of the chromosome can be ignored. Moreover, there can be more than a 10-fold difference in usage between repair sites located only 10 kb apart. Interestingly, arms of a given chromosome are less accessible to one another than to other chromosomes. Altogether, these results indicate that the accessibility between genomic sites in the human genome during DSB repair is specific and conserved in a cell population.     

Diversity of the damage recognition step in the global genomic nucleotide excision repair in vitro

The XPC-HR23B complex, a mammalian factor specifically involved in global genomic nucleotide excision repair (NER) has been shown to bind various forms of damaged DNA and initiate DNA repair in cell-free reactions. To characterize the binding specificity of this factor in more detail, a method based on immunoprecipitation was developed to assess the relative affinity of XPC-HR23B for defined lesions on DNA. Here we show that XPC-HR23B preferentially binds to UV-induced (6-4) photoproducts (6-4PPs) as well as to cholesterol, but not to the cyclobutane pyrimidine dimer (CPD), 8-oxoguanine (8-oxo-G), O6-methylguanine (O6-Me-G), or a single mismatch. Human whole cell extracts could efficiently excise 6-4PPs and cholesterol in an XPC-HR23B-dependent manner, but not 8-oxo-G, O6-Me-G or mismatches. Thus, there was good correlation between the binding specificity of XPC-HR23B for certain types of lesion and the ability of human cell extracts to excise these lesions, supporting the model that XPC-HR23B initiates global genomic NER. Although, XPC-HR23B does not preferentially bind to CPDs, the excision of CPDs in human whole cell extracts was found to be absolutely dependent on XPC-HR23B, in agreement with the in vivo observation that CPDs are not removed from the global genome in XP-C mutant cells. These results suggest that, in addition to the excision repair pathway initiated by XPC-HR23B, there exists another sub-pathway for the global genomic NER that still requires XPC-HR23B but is not initiated by XPC-HR23B. Possible mechanisms will be discussed.