Mouse anti-Human RAD52 monoclonal antibody, clone 6I0

Tumour repressor p53 isoform Delta 133p53 is a target gene of p53 and an antagonist of p53-mediated apoptotic activity. We recently demonstrated that Delta 133p53 promotes DNA double-strand break (DSB) repair by upregulating transcription of the repair genes RADS1, LIG4 and RAD52 in a p53-independent manner. However, Delta 133p53 lacks the trans activation domain of full-length p53, and the mechanism by which it exerts transcriptional activity independently of full-length p53 remains unclear. In this report, we describe the accumulation of high levels of both Delta 133p53 and p73 (a p53 family member) at 24 h post gamma-irradiation (hpi). Delta 133p53 can form a complex with p73 upon gamma-irradiation. The co-expression of Delta 133p53 and p73, but not either protein alone, can significantly promote DNA DSB repair mechanisms, including homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA). p73 and Delta 133p53 act synergistically to promote the expression of RADS1, LIG4 and RAD52 by joining together to bind to region containing a Delta 133p53-responsive element (RE) and a p73-RE in the promoters of all three repair genes. In addition to its accumulation at 24 hpi, p73 protein expression also peaks at 4 hpi. The depletion of p73 not only reduces early-stage apoptotic frequency (4-6 hpi), but also significantly increases later-stage DNA DSB accumulation (48 hpi), leading to cell cycle arrest in the G2 phase and, ultimately, cell senescence. In summary, the apoptotic regulator p73 also coordinates with Delta 133p53 to promote DNA DSB repair, and the loss of function of p73 in DNA DSB repair may underlie spontaneous and carcinogen-induced tumorigenesis in p73 knockout mice.

The function of the replication clamp loaders in the semi-conservative telomere replication and their relationship to telomerase-and recombination mechanisms of telomere addition remains ambiguous. We have investigated the variant clamp loader Ctf18 RFC (Replication Factor C). To understand the role of Ctf18 at the telomere, we first investigated genetic interactions after loss of Ctf18 and TLC1 (the yeast telomerase RNA). We find that the tlc1 Delta ctf18 Delta double mutant confers a rapid >1000-fold decrease in viability. The rate of loss was similar to the kinetics of cell death in rad52 Delta tlc1 Delta cells. However, the Ctf18 pathway is distinct from Rad52, required for the repair of DSBs, as demonstrated by the synthetic lethality of rad52 Delta tlc1 Delta ctf18 Delta triple mutants. These data suggest that each mutant elicits non-redundant defects acting on the same substrate. Second, interactions of the yeast hyper-recombinational mutant, mre11A470T, with ctf18 Delta confer a synergistic cold sensitivity. The phenotype of these double mutants ultimately results in telomere loss and the generation of recombinational survivors. We observed a similar synergism between single mutants that led to hypersensitivity to the DNA alkylating agent, methane methyl sulphonate (MMS), the replication fork inhibitor hydroxyurea (HU), and to a failure to separate telomeres of sister chromatids. Hence, ctf18 Delta and mre11A470T act in different pathways on telomere substrates for multiple phenotypes. The mre11A470T cells also displayed a DNA damage response (DDR) at 15 degrees C but not at 30 degrees C while ctf18 Delta mutants conferred a constitutive DDR activity. Both the 15 degrees C DDR pattern and growth rate were reversible at 30 degrees C and displayed telomerase activity in vivo. We hypothesize that Ctf18 confers protection against stalling and/or breaks at the replication fork in cells that either lack, or are compromised for, telomerase activity. This Ctf18-based function is likely to contribute another level to telomere size homeostasis.