Telomere maintenance requires the RAD51D recombination/repair protein.

The five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are required in mammalian cells for normal levels of genetic recombination and resistance to DNA-damaging agents. We report here that RAD51D is also involved in telomere maintenance. Using immunofluorescence labeling, electron microscopy, and chromatin immunoprecipitation assays, RAD51D was shown to localize to the telomeres of both meiotic and somatic cells. Telomerase-positive Rad51d(-/-) Trp53(-/-) primary mouse embryonic fibroblasts (MEFs) exhibited telomeric DNA repeat shortening compared to Trp53(-/-) or wild-type MEFs. Moreover, elevated levels of chromosomal aberrations were detected, including telomeric end-to-end fusions, a signature of telomere dysfunction. Inhibition of RAD51D synthesis in telomerase-negative immortalized human cells by siRNA also resulted in telomere erosion and chromosome fusion. We conclude that RAD51D plays a dual cellular role in both the repair of DNA double-strand breaks and telomere protection against attrition and fusion

Sister chromatid exchanges occur in G2-irradiated cells

DNA double-strand breaks (DSBs) are arguably the most important lesions induced by ionizing radiation (IR) since unrepaired or misrepaired DSBs can lead to chromosomal aberrations and cell death. The two major pathways to repair IR-induced DSBs are non-homologous end-joining (NHEJ) and homologous recombination (HR). Perhaps surprisingly, NHEJ represents the predominant pathway in the G1 and G2 phases of the cell cycle, but HR also contributes and repairs a subset of IR-induced DSBs in G2. Following S-phase-dependent genotoxins, HR events give rise to sister chromatid exchanges (SCEs), which can be detected cytogenetically in mitosis. Here, we describe that HR occurring in G2-irradiated cells also generates SCEs in ∼50% of HR events. Since HR of IR-induced DSBs in G2 is a slow process, SCE formation in G2-irradiated cells requires several hours. During this time, irradiated S-phase cells can also reach mitosis, which has contributed to the widely held belief that SCEs form only during S phase. We describe procedures to measure SCEs exclusively in G2-irradiated cells and provide evidence that following IR cells do not need to progress through S phase in order to form SCEs

Sws1 is a conserved regulator of homologous recombination in eukaryotic cells

Rad52-dependent homologous recombination (HR) is regulated by the antirecombinase activities of Srs2 and Rqh1/Sgs1 DNA helicases in fission yeast and budding yeast. Functional analysis of Srs2 in Schizosaccharomyces pombe led us to the discovery of Sws1, a novel HR protein with a SWIM-type Zn finger. Inactivation of Sws1 suppresses the genotoxic sensitivity of srs2Δ and rqh1Δ mutants and rescues the inviability of srs2Δ rqh1Δ cells. Sws1 functions at an early step of recombination in a pro-recombinogenic complex with Rlp1 and Rdl1, two RecA-like proteins that are most closely related to the human Rad51 paralogs XRCC2 and RAD51D, respectively. This finding indicates that the XRCC2–RAD51D complex is conserved in lower eukaryotes. A SWS1 homolog exists in human cells. It associates with RAD51D and ablating its expression reduces the number of RAD51 foci. These studies unveil a conserved pathway for the initiation and control of HR in eukaryotic cells

Reduced FANCD2 influences spontaneous SCE and RAD51 foci formation in uveal melanoma and Fanconi anaemia

Uveal melanoma (UM) is unique among cancers in displaying reduced endogenous levels of sister chromatid exchange (SCE). Here we demonstrate that FANCD2 expression is reduced in UM and that ectopic expression of FANCD2 increased SCE. Similarly, FANCD2-deficient fibroblasts (PD20) derived from Fanconi anaemia patients displayed reduced spontaneous SCE formation relative to their FANCD2-complemented counterparts, suggesting that this observation is not specific to UM. In addition, spontaneous RAD51 foci were reduced in UM and PD20 cells compared with FANCD2-proficient cells. This is consistent with a model where spontaneous SCEs are the end product of endogenous recombination events and implicates FANCD2 in the promotion of recombination-mediated repair of endogenous DNA damage and in SCE formation during normal DNA replication. In both UM and PD20 cells, low SCE was reversed by inhibiting DNA-PKcs (DNA-dependent protein kinase, catalytic subunit). Finally, we demonstrate that both PD20 and UM are sensitive to acetaldehyde, supporting a role for FANCD2 in repair of lesions induced by such endogenous metabolites. Together, these data suggest FANCD2 may promote spontaneous SCE by influencing which double-strand break repair pathway predominates during normal S-phase progression

DNA-damage repair; the good, the bad, and the ugly

Organisms have developed several DNA-repair pathways as well as DNA-damage checkpoints to cope with the frequent challenge of endogenous and exogenous DNA insults. In the absence or impairment of such repair or checkpoint mechanisms, the genomic integrity of the organism is often compromised. This review will focus on the functional consequences of impaired DNA-repair pathways. Although each pathway is addressed individually, it is essential to note that cross talk exists between repair pathways, and that there are instances in which a DNA-repair protein is involved in more than one pathway. It is also important to integrate DNA-repair process with DNA-damage checkpoints and cell survival, to gain a better understanding of the consequences of compromised DNA repair at both cellular and organismic levels. Functional consequences associated with impaired DNA repair include embryonic lethality, shortened life span, rapid ageing, impaired growth, and a variety of syndromes, including a pronounced manifestation of cancer