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.

SummaryTo improve replication fidelity, mismatch repair (MMR) must detect non-Watson-Crick base pairs and direct their repair to the nascent DNA strand. Eukaryotic MMR in vitro requires pre-existing strand discontinuities for initiation; consequently, it has been postulated that MMR in vivo initiates at Okazaki fragment termini in the lagging strand and at nicks generated in the leading strand by the mismatch-activated MLH1/PMS2 endonuclease. We now show that a single ribonucleotide in the vicinity of a mismatch can act as an initiation site for MMR in human cell extracts and that MMR activation in this system is dependent on RNase H2. As loss of RNase H2 in S.cerevisiae results in a mild MMR defect that is reflected in increased mutagenesis, MMR in vivo might also initiate at RNase H2-generated nicks. We therefore propose that ribonucleotides misincoporated during DNA replication serve as physiological markers of the nascent DNA strand.

SummaryThe LEM domain (for lamina-associated polypeptide, emerin, MAN1 domain) defines a group of nuclear proteins that bind chromatin through interaction of the LEM motif with the conserved DNA crosslinking protein, barrier-to-autointegration factor (BAF). Here, we describe a LEM protein annotated in databases as 'Ankyrin repeat and LEM domain-containing protein 1' (Ankle1). We show that Ankle1 is conserved in metazoans and contains a unique C-terminal GIY-YIG motif that confers endonuclease activity in vitro and in vivo. In mammals, Ankle1 is predominantly expressed in hematopoietic tissues. Although most characterized LEM proteins are components of the inner nuclear membrane, ectopic Ankle1 shuttles between cytoplasm and nucleus. Ankle1 enriched in the nucleoplasm induces DNA cleavage and DNA damage response. This activity requires both the catalytic C-terminal GIY-YIG domain and the LEM motif, which binds chromatin via BAF. Hence, Ankle1 is an unusual LEM protein with a GIY-YIG-type endonuclease activity in higher eukaryotes.

The damage to DNA caused by ultraviolet B radiation (280-320 nm) contributes significantly to development of sunlight-induced skin cancers. The susceptibility of mice to ultraviolet B-induced skin carcinogenesis is increased by an inhibitor of the DNA damage-activated nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP), hence PARP activation is likely to be associated with cellular responses that suppress carcinogenesis. To understand the role of activated PARP in these cellular functions, we need to first clearly identify the cause of PARP activation in ultraviolet B-irradiated cells. Ultraviolet B, like ultraviolet C, causes direct DNA damage of cyclobutane pyrimidine dimer and 6, 4-photoproduct types, which are subjected to the nucleotide excision repair. Moreover, ultraviolet B also causes oxidative DNA damage, which is subjected to base excision repair. To identify which of these two types of DNA damage activates PARP, we examined mechanism of early PARP activation in mouse fibroblasts exposed to ultraviolet B and C radiations. The ultraviolet B-irradiated cells rapidly activated PARP in two distinct phases, initially within the first 5 minutes and later between 60-120 minutes, whereas ultraviolet C-irradiated cells showed only the immediate PARP activation. Using antioxidants, local irradiation, chromatin immunoprecipitation and in vitro PARP assays, we identified that ultraviolet radiation-induced direct DNA damage, such as thymine dimers, cause the initial PARP activation, whereas ultraviolet B-induced oxidative damage cause the second PARP activation. Our results suggest that cells can selectively activate PARP for participation in different cellular responses associated with different DNA lesions.