ATM (ataxia telangiectasia-mutated) and ATR (ATM-Rad3-related) are proximal checkpoint kinases that regulate DNA damage response (DDR). Identification and characterization of ATM/ATR substrates hold the keys for the understanding of DDR. Few techniques are available to identify protein kinase substrates. Here, we screened for potential ATM/ATR substrates using phospho-specific antibodies against known ATM/ ATR substrates. We identified proteins cross-reacting to phospho-specific antibodies in response to DNA damage by mass spectrometry. We validated a subset of the candidate substrates to be phosphorylated in an ATM/ATR-dependent manner in vivo. Combining with a functional checkpoint screen, we identified proteins that belong to the ubiquitin-proteasome system (UPS) to be required in mammalian DNA damage checkpoint control, particularly the G 1 cell cycle checkpoint, thus revealing protein ubiquitylation as an important regulatory mechanism downstream of ATM/ATR activation for checkpoint control.

DNA single-strand break repair (SSBR) is important for maintaining genome stability and homeostasis. The current SSBR model derived from an in vitro-reconstituted reaction suggests that the SSBR complex mediated by X-ray repair cross-complementing protein 1 (XRCC1) is assembled sequentially at the site of damage. In this study, we provide biochemical data to demonstrate that two preformed XRCC1 protein complexes exist in cycling HeLa cells. One complex contains known enzymes that are important for SSBR, including DNA ligase 3 (DNL3), polynucleotide kinase 3-phosphatase, and polymerase ␤; the other is a new complex that contains DNL3 and the ataxia with oculomotor apraxia type 1 (AOA) gene product aprataxin. We report the characterization of the new XRCC1 complex. XRCC1 is phosphorylated in vivo and in vitro by CK2, and CK2 phosphorylation of XRCC1 on S518, T519, and T523 largely determines aprataxin binding to XRCC1 though its FHA domain. An acute loss of aprataxin by small interfering RNA renders HeLa cells sensitive to methyl methanesulfonate treatment by a mechanism of shortened half-life of XRCC1. Thus, aprataxin plays a role to maintain the steady-state protein level of XRCC1. Collectively, these data provide insights into the SSBR molecular machinery in the cell and point to the involvement of aprataxin in SSBR, thus linking SSBR to the neurological disease AOA.

Adenovirus type 7 caused a high proportion of severe infections.

In unstressed cells, the tumor suppressor p53 is maintained at low levels by ubiquitin-mediated proteolysis mainly through Mdm2. In response to DNA damage, p53 is stabilized and becomes activated to turn on transcriptional programs that are essential for cell cycle arrest and apoptosis. Activation of p53 leads to accumulation of Mdm2 protein, a direct transcriptional target of p53. It is not understood how p53 is protected from degradation when Mdm2 is upregulated. Here we report that p53 stabilization in the late phase after ionizing radiation correlates with active ubiquitination. We found that an E3 ubiquitin ligase RFWD3 (RNF201/FLJ10520) forms a complex with Mdm2 and p53 to synergistically ubiquitinate p53 and is required to stabilize p53 in the late response to DNA damage. This process is regulated by the DNA damage checkpoint, because RFWD3 is phosphorylated by ATM/ATR kinases and the phosphorylation mutant fails to stimulate p53 ubiquitination. In vitro experiments suggest that RFWD3 is a p53 E3 ubiquitin ligase and that RFWD3-Mdm2 complex restricts the polyubiquitination of p53 by Mdm2. Our study identifies RFWD3 as a positive regulator of p53 stability when the G 1 cell cycle checkpoint is activated and provides an explanation for how p53 is protected from degradation in the presence of high levels of Mdm2.T he p53 tumor suppressor is a key regulator of cell cycle arrest and apoptosis in response to genotoxic stress (1-3). The G 1 cell cycle checkpoint is operated through the p53-dependent transcriptional response (4). Accumulation of the p53 target gene product p21 WAF1∕CIP1 after DNA damage to a suprathreshold level, capable of blocking the G 1 -S promoting cyclinE/Cdk2 activity, may require several hours and is responsible for the sustained G 1 arrest (5).p53 is primarily regulated at the level of protein stability (6, 7). At least five E3 ligases (E6-AP, Mdm2, Arf-BP1, COP1, and Pirh2) have been identified to mediate ubiquitin-dependent proteasomal degradation of p53. Each of the E3 ligases is capable of building K48-linked polyubiquitin chains on p53, which are recognized by 26S proteasome for degradation. In response to DNA damage, it is thought that such polyubiquitination is inhibited to stabilize p53 protein. Mdm2 is the major p53 E3 ligase, because embryonic lethality of Mdm2-knockout mice caused by p53-induced apoptosis is rescued by deletion of p53 (8, 9).One paradox exists in the Mdm2 axis for p53 stabilization. Because Mdm2 is a p53 transcription target, stabilization of p53 leads to up-regulation of Mdm2 (10), which in turn should degrade p53, but p53 is maintained at high levels when the G 1 checkpoint is active. It was proposed that DNA damage destabilizes Mdm2 by a mechanism involving phosphorylation by ATM/ ATR and increased Mdm2 turnover. Thus, accelerated Mdm2 ubiquitination shortens its half-life, suppressing its activity towards p53 (11). Posttranslational modification of p53 is another important mechanism for its regulation (6). p53 modification by phosphorylation, acetylation, ...