ATM creates a veil of transcriptional silence

The ATM kinase orchestrates diverse responses to DNA damage. By simultaneously monitoring transcription and DNA-damage responses in single cells, Shanbhag et al. (2010) now uncover a role of ATM in preventing transcription near DNA double-strand breaks. © 2010 Elsevier Inc.postprin

Role of ATM signaling in PALB2-dependent DNA damage responses

Poster presentation - Theme 1: Cell biologyMutations of the PALB2 gene lead to a number of hereditary cancer-predisposing syndromes, including Fanconi anemia and hereditary breast and ovarian cancer syndrome. Originally identified as a core DNA repair factor, emerging evidence now implicates PALB2 in cell cycle checkpoint control, DNA replication, oxidative stress regulation and transcription, highlighting the multi-functionality of the tumor suppressor. Notably, mechanistically how its expanding repertoire of functions are orchestrated remains ...postprin

Molecular architecture of the Ub-PCNA/Pol η complex bound to DNA

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The Ubiquitin Specific Protease USP34 promotes ubiquitin signaling at DNA double-strand breaks

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Distinct versus overlapping functions of MDC1 and 53BP1 in DNA damage response and tumorigenesis

The importance of the DNA damage response (DDR) pathway in development, genomic stability, and tumor suppression is well recognized. Although 53BP1 and MDC1 have been recently identified as critical upstream mediators in the cellular response to DNA double-strand breaks, their relative hierarchy in the ataxia telangiectasia mutated (ATM) signaling cascade remains controversial. To investigate the divergent and potentially overlapping functions of MDC1 and 53BP1 in the ATM response pathway, we generated mice deficient for both genes. Unexpectedly, the loss of both MDC1 and 53BP1 neither significantly increases the severity of defects in DDR nor increases tumor incidence compared with the loss of MDC1 alone. We additionally show that MDC1 regulates 53BP1 foci formation and phosphorylation in response to DNA damage. These results suggest that MDC1 functions as an upstream regulator of 53BP1 in the DDR pathway and in tumor suppression. © 2008 Minter-Dykhouse et al. The Rockefeller University Press.published_or_final_versio

An E2-guided E3 Screen Identifies the RNF17-UBE2U Pair as Regulator of the Radiosensitivity, Immunodeficiency, Dysmorphic Features, and Learning Difficulties (RIDDLE) Syndrome Protein RNF168

Protein ubiquitination has emerged as a pivotal regulatory reaction that promotes cellular responses to DNA damage. With a goal to delineate the DNA damage signal transduction cascade, we systematically analyzed the human E2 ubiquitin- and ubiquitin-like-conjugating enzymes for their ability to mobilize the DNA damage marker 53BP1 onto ionizing radiation-induced DNA double strand breaks. An RNAi-based screen identified UBE2U as a candidate regulator of chromatin responses at double strand breaks. Further mining of the UBE2U interactome uncovered its cognate E3 RNF17 as a novel factor that, via the radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties (RIDDLE) syndrome protein RNF168, enforces DNA damage responses. Our screen allowed us to uncover new players in the mammalian DNA damage response and highlights the instrumental roles of ubiquitin machineries in promoting cell responses to genotoxic stress.published_or_final_versio

Cell death caused by single-stranded oligodeoxynucleotide-mediated targeted genomic sequence modification

Targeted gene repair directed by single-stranded oligodeoxynucleotides (ssODNs) offers a promising tool for biotechnology and gene therapy. However, the methodology is currently limited by its low frequency of repair events, variability, and low viability of "corrected" cells. In this study, we showed that during ssODN-mediated gene repair reaction, a significant population of corrected cells failed to divide, and were much more prone to undergo apoptosis, as marked by processing of caspases and PARP-1. In addition, we found that apoptotic cell death triggered by ssODN-mediated gene repair was largely independent of the ATM/ATR kinase. Furthermore, we examined the potential involvement of the mismatch repair (MMR) proteins in this "correction reaction-induced" cell death. Result showed that while defective MMR greatly enhanced the efficiency of gene correction, compromising the MMR system did not yield any viable corrected clone, indicating that the MMR machinery, although plays a critical role in determining ssODN-directed repair, was not involved in the observed cellular genotoxic responses. © 2009. Mary Ann Liebert, Inc.published_or_final_versio

TRAIP regulates replication fork recovery and progression via PCNA

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Differential regulation of RNF8-mediated Lys48- and Lys63-based poly-ubiquitylation

Pairing of a given E3 ubiquitin ligase with different E2s allows synthesis of ubiquitin conjugates of different topologies. While this phenomenon contributes to functional diversity, it remains largely unknown how a single E3 ubiquitin ligase recognizes multiple E2s, and whether identical structural requirements determine their respective interactions. The E3 ubiquitin ligase RNF8 that plays a critically important role in transducing DNA damage signals, interacts with E2s UBCH8 and UBC13, and catalyzes both K48- and K63-linked ubiquitin chains. Interestingly, we report here that a single-point mutation (I405A) on the RNF8 polypeptide uncouples its ability in catalyzing K48- and K63-linked ubiquitin chain formation. Accordingly, while RNF8 interacted with E2s UBCH8 and UBC13, its I405A mutation selectively disrupted its functional interaction with UBCH8, and impaired K48-based poly-ubiquitylation reactions. In contrast, RNF8 I405A preserved its interaction with UBC13, synthesized K63-linked ubiquitin chains, and assembled BRCA1 and 53BP1 at sites of DNA breaks. Together, our data suggest that RNF8 regulates K48- and K63-linked poly-ubiquitylation via differential RING-dependent interactions with its E2s UBCH8 and UBC13, respectively.published_or_final_versio

Impact of G 2 checkpoint defect on centromeric instability

Centromeric instability is characterized by dynamic formation of centromeric breaks, deletions, isochromosomes and translocations, which are commonly observed in cancer. So far, however, the mechanisms of centromeric instability in cancer cells are still poorly understood. In this study, we tested the hypothesis that G 2 checkpoint defect promotes centromeric instability. Our observations from multiple approaches consistently support this hypothesis. We found that overexpression of cyclin B1, one of the pivotal genes driving G 2 to M phase transition, impaired G 2 checkpoint and promoted the formation of centromeric aberrations in telomerase-immortalized cell lines. Conversely, centromeric instability in cancer cells was ameliorated through reinforcement of G 2 checkpoint by cyclin B1 knockdown. Remarkably, treatment with KU55933 for only 2.5 h, which abrogated G 2 checkpoint, was sufficient to produce centromeric aberrations. Moreover, centromeric aberrations constituted the major form of structural abnormalities in G 2 checkpoint-defective ataxia telangiectasia cells. Statistical analysis showed that the frequencies of centromeric aberrations in G 2 checkpoint-defective cells were always significantly overrepresented compared with random assumption. As there are multiple pathways leading to G 2 checkpoint defect, our finding offers a broad explanation for the common occurrence of centromeric aberrations in cancer cells. © 2011 Macmillan Publishers Limited All rights reserved.postprin