Eukaryotic cells have evolved to use complex pathways for DNA damage signaling and repair to maintain genomic integrity. RNF168 is a novel E3 ligase that functions downstream of ATM,γ-H2A.X, MDC1, and RNF8. It has been shown to ubiquitylate histone H2A and to facilitate the recruitment of other DNA damage response proteins, including 53BP1, to sites of DNA break. In addition, RNF168 mutations have been causally linked to the human RIDDLE syndrome. In this study, we report that Rnf168−/− mice are immunodeficient and exhibit increased radiosensitivity. Rnf168−/− males suffer from impaired spermatogenesis in an age-dependent manner. Interestingly, in contrast to H2a.x−/−, Mdc1−/−, and Rnf8−/− cells, transient recruitment of 53bp1 to DNA double-strand breaks was abolished in Rnf168−/− cells. Remarkably, similar to 53bp1 inactivation, but different from H2a.x deficiency, inactivation of Rnf168 impairs long-range V(D)J recombination in thymocytes and results in long insertions at the class-switch junctions of B-cells. Loss of Rnf168 increases genomic instability and synergizes with p53 inactivation in promoting tumorigenesis. Our data reveal the important physiological functions of Rnf168 and support its role in both γ-H2a.x-Mdc1-Rnf8-dependent and -independent signaling pathways of DNA double-strand breaks. These results highlight a central role for RNF168 in the hierarchical network of DNA break signaling that maintains genomic integrity and suppresses cancer development in mammals.

The essential role of dietary micronutrients for genome stability is well documented, yet the effect of folate deficiency or excess on telomeres is not known. Accordingly, human WIL2-NS cells were maintained in medium containing 30, 300, or 3,000 nmol/L folic acid (FA) for 42 days to test the hypothesis that chronic folate deficiency would cause telomere shortening and dysfunction. After 14 days, telomere length (TL) in FA-deficient (30 nmol/L) cultures was 26% longer than that of 3,000 nmol/L FA cultures; however, this was followed by rapid telomere attrition over the subsequent 28 days (P trend, P < 0.0001); both long and short telomere status was positively correlated with biomarkers of chromosome instability (P 0.003) and mitotic dysfunction (P ¼ 0.01), measured by the cytokinesis-block micronucleus cytome (CBMN-cyt) assay. The early increase in TL was associated with FA-deficiency-induced global DNA hypomethylation (P ¼ 0.05), with an effect size similar to that induced by the DNA methyltransferase inhibitor, 5-aza-2 0 -deoxycytidine. Quantitative PCR analysis indicated a negative association between FA concentration and uracil incorporation into telomeric DNA (r ¼ À0.47, P ¼ 0.1), suggesting a possible plausible mechanism for uracil as a cause of folate deficiency-induced telomere dysfunction or deletion. Peptide nucleic acid-FISH (PNA-FISH) analysis showed that FA deficiency resulted in 60% of micronuclei containing acentric terminal fragments, an observation consistent with the 3-fold increase in terminal deletions (P ¼ 0.0001). Together, these results demonstrate the impact of folate deficiency on biomarkers of telomere maintenance and integrity, and provide evidence that dysfunctional long telomeres may be as important as critically short telomeres as a cause of chromosomal instability. Cancer Prev Res; 7(1); 128-38. Ó2013 AACR.