No abstract

During mild replication stress provoked by low dose aphidicolin (APH) treatment, the key Fanconi anemia protein FANCD2 accumulates on common fragile sites, observed as sister foci, and protects genome stability. To gain further insights into FANCD2 function and its regulatory mechanisms, we examined the genome-wide chromatin localization of FANCD2 in this setting by ChIP-seq analysis. We found that FANCD2 mostly accumulates in the central regions of a set of large transcribed genes that were extensively overlapped with known CFS. Consistent with previous studies, we found that this FANCD2 retention is R-loop-dependent. However, FANCD2 monoubiquitination and RPA foci formation were still induced in cells depleted of R-loops. Interestingly, we detected increased Proximal Ligation Assay dots between FANCD2 and R-loops following APH treatment, which was suppressed by transcriptional inhibition. Collectively, our data suggested that R-loops are required to retain FANCD2 in chromatin at the middle intronic region of large genes, while the replication stress-induced upstream events leading to the FA pathway activation are not triggered by R-loops.

Amyotrophic lateral sclerosis (ALS)5 is a progressive, typically fatal, neurodegenerative disease that targets motor neurons (1). The cumulative lifetime risk for ALS is ϳ1 in 1000, which is comparable with the occurrence rate of multiple sclerosis (1). However, only about 3,000 cases of ALS are observed at any given time in the United States, because of the low fiveyear median survival rate (20%) for this disease. There are currently no effective treatments to halt the course of ALS.Approximately 10% of ALS cases, termed familial ALS (fALS), have a clear genetic link. Dominant mutations in the Cu,Zn-superoxide dismutase 1 (SOD1) are responsible for ϳ20% of fALS (2). Well over 100 different disease-associated mutations in SOD1 have been identified in humans (3). The mutations occur throughout the SOD1 open reading frame and do not alter the catalytic properties of the SOD1 enzyme per se (4). Instead, mutant SOD1 proteins are thought to acquire an abnormal fold that confers a toxic gain-of-function activity (4). Multiple cellular processes, including mitochondrial function, axon transport, and glutamate transporter function are adversely affected by toxic SOD1 mutants (5-10). Remarkably, mouse models of SOD1-induced ALS have shown that it is not a neuron-autonomous disease; expression of toxic SOD1 mutants in non-neuronal glial cells is sufficient to induce neuron pathology, whereas expression of toxic SOD1 mutants in motor neurons is insufficient for full expression of the disease phenotype (11,12). The SOD1 mouse model has been used to test candidate ALS therapeutics, including gene therapies directed toward silencing toxic SOD1 alleles in fALS (13)(14)(15).Sporadic ALS (sALS), representing 90 -95% of all ALS cases, occurs in the absence of a family history of disease, but follows a clinical course that is similar to SOD1 ALS. A hallmark of sALS is the presence of ubiquitin (Ub)-positive cytosolic aggregates in degenerating motor neurons (16). Recently, Neumann et al. (17) demonstrated that the 43-kDa human immunodeficiency virus transactivating region DNA-binding domain protein (TDP-43) is a major protein component of ubiquitin-positive aggregates in patients with sALS or ubiquitin-positive fronto-temporal lobular dementia. First identified as a binding factor of the long-terminal repeat region of the human immunodeficiency virus genome, TDP-43 is a nuclear RNA-binding protein that regulates splicing of the cystic fibrosis transmembrane conductance regulator mRNA (18 -20). Inactivation of TDP-43 through RNA interference caused enhanced transcription of cyclin-dependent kinase 6, hyperphosphorylation of the

The DNA damage surveillance network orchestrates cellular responses to DNA damage through the recruitment of DNA damage-signaling molecules to DNA damage sites and the concomitant activation of protein phosphorylation cascades controlled by the ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) kinases. Activation of ATM/ATR triggers cell cycle checkpoint activation and adaptive responses to DNA damage. Recent studies suggest that protein ubiquitylation or degradation plays an important role in the DNA damage response. In this study, we examined the potential role of the proteasome in checkpoint activation and ATM/ATR signaling in response to UV light-induced DNA damage. HeLa cells treated with the proteasome inhibitor MG-132 showed delayed phosphorylation of ATM substrates in response to UV light. UV light-induced phosphorylation of 53BP1, as well as its recruitment to DNA damage foci, was strongly suppressed by proteasome inhibition, whereas the recruitment of upstream regulators of 53BP1, including MDC1 and H2AX, was unaffected. The ubiquitin-protein isopeptide ligase RNF8 was critical for 53BP1 focus targeting and phosphorylation in ionizing radiation-damaged cells, whereas UV light-induced 53BP1 phosphorylation and targeting exhibited partial dependence on RNF8 and the ubiquitin-conjugating enzyme UBC13. Suppression of RNF8 or UBC13 also led to subtle defects in UV light-induced G 2 /M checkpoint activation. These findings are consistent with a model in which RNF8 ubiquitylation pathways are essential for 53BP1 regulation in response to ionizing radiation, whereas RNF8-independent pathways contribute to 53BP1 targeting and phosphorylation in response to UV light and potentially other forms of DNA replication stress.DNA-damaging stimuli elicit highly conserved responses in eukaryotic cells that are required for the maintenance of genomic integrity and organismal viability. Protein kinase cascades are central to the DNA damage-signaling paradigm, and members of the phosphoinositide 3-kinase-related kinase gene superfamily play particularly important roles as apical DNA damage response regulators. Two functionally related members of the phosphoinositide 3-kinase-related kinase family, the ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) kinases, initiate overlapping signaling pathways in response to distinct types of genetic lesions. ATM is principally activated by ionizing radiation (IR) 2 and other agents that induce DNA double-strand breaks, whereas ATR responds to pauses in DNA replication that transiently expose single-strand DNA (1). Once activated, ATM and ATR phosphorylate and activate the CHK2 and CHK1 protein kinases, respectively, which in turn promote cell cycle checkpoint arrest through inhibition of CDC25 family phosphatases (2-4). ATM and ATR phosphorylate numerous other substrates, including p53 and BRCA1, which participate in DNA metabolism. The regulatory reach of ATM and ATR extends to many other physiologic processes, and recent studies suggest that the true numb...

Replication protein A2 (RPA2), a component of the RPA heterotrimer, is hyperphosphorylated and forms nuclear foci in response to camptothecin (CPT) that directly induces replication‐mediated DNA double‐strand breaks (DSBs). Ataxia‐telangiectasia mutated and Rad3‐related kinase (ATR) and DNA‐dependent protein kinase (DNA‐PK) are activated by CPT, and RPA2 is hyperphosphorylated in a DNA‐PK‐dependent manner. To distinguish the roles of phosphatidylinositol 3‐kinase‐related protein kinases including DNA‐PK, ataxia‐telangiectasia mutated (ATM), and ATR, in the response to replication‐mediated DSBs, we analyzed RPA2 focus formation and hyperphosphorylation during exposure to CPT. ATR knock‐down with siRNA suppressed CPT‐induced RPA2 hyperphosphorylation and focus formation. CPT‐induced RPA2 focus formation was normally observed in DNA‐PK‐ or ATM‐deficient cells. Comparison between CPT and hydroxyurea (HU) indirectly inducing DSBs showed that RPA2 hyperphosphorylation is DNA‐PK‐dependent in CPT‐treated cells and DNA‐PK‐independent in HU‐treated cells. Although RPA2 foci rapidly formed in response to HU and CPT, the RPA2 hyperphosphorylation in HU‐treated cells occurred later than in the CPT‐treated cells, indicating that the DNA‐PK dependency of RPA2 hyperphosphorylation is likely to be related to the mode of DSB induction. These results suggest that DNA‐PK is responsible for the RPA2 hyperphosphorylation following ATR‐dependent RPA2 focus formation in response to replication‐mediated DSBs directly induced by CPT.

Background:It is unclear how DNA-damaging agents target cancer cells over normal somatic cells. Results: Arf/p53-dependent down-regulation of H2AX enables normal cells to survive after DNA damage. Conclusion: Transformed cells, which harbor mutations in either Arf or p53, are more sensitive to DNA-damaging agents. Significance: Cellular transformation renders cells more susceptible to some DNA-damaging agents.

Temporal regulation of microtubule dynamics is essential for proper progression of mitosis and control of microtubule plus-end tracking proteins by phosphorylation is an essential component of this regulation. Here we show that Aurora B and CDK1 phosphorylate microtubule end-binding protein 2 (EB2) at multiple sites within the amino terminus and a cluster of serine/threonine residues in the linker connecting the calponin homology and end-binding homology domains. EB2 phosphorylation, which is strictly associated with mitotic entry and progression, reduces the binding affinity of EB2 for microtubules. Expression of non-phosphorylatable EB2 induces stable kinetochore microtubule dynamics and delays formation of bipolar metaphase plates in a microtubule binding-dependent manner, and leads to aneuploidy even in unperturbed mitosis. We propose that Aurora B and CDK1 temporally regulate the binding affinity of EB2 for microtubules, thereby ensuring kinetochore microtubule dynamics, proper mitotic progression and genome stability.

The topoisomerase I (TopI) 2 poison camptothecin (CPT) and its clinically relevant derivatives, topotecan and irinotecan, have been intensively studies for their tumoricidal properties. The molecular target of CPT is TopI, an enzyme that mediates the relaxation of supercoiled DNA (1). This is achieved through the transient introduction of a DNA single-strand break that permits the rotational relaxation of double-stranded DNA. The TopI reaction cycle involves the formation of a transient phosphotyrosyl bond between Tyr 723 of the enzyme active site and a 3Ј DNA end. CPT stabilizes covalent TopI-DNA cleavage complexes (TopI-cc), which are converted into DNA doublestrand breaks (DSBs) upon encountering active DNA replication forks (1).The signaling and repair of CPT-mediated damage have been the focus of intensive research. CPT-induced DSBs possess a single DNA double-strand end (DSE) that is generally not an efficient substrate for nonhomologous end joining DSB repair, which is mediated by DNA-dependent protein kinase (DNA-PK) composed of DNA-PKcs and Ku protein. Instead, CPTinduced DSEs are repaired by homologous recombination (HR) repair, which utilizes a sister chromatid and primes restart of the obstructed DNA replication fork (2, 3). CPT-induced DSEs strongly activate cell cycle checkpoint pathways downstream of the ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) protein kinases. CPT-induced damage in S phase activates the ATR-Chk1 pathway that mediates S phase arrest (4). Consistent with these important S phase functions, ATRor Chk1-deficient cells are exquisitely sensitive to TopI poisons (4, 5).In addition to replication-mediated DNA damage, TopI-cc pose a block to processive RNA polymerase complexes, and the collision of RNA polymerase with TopI-cc results in transcription-mediated DNA damage (6). Although the mechanisms of transcription-coupled damage and signaling are not well understood, recent studies suggest a new role for the ATM protein kinase. CPT-induced activation of ATM is suppressed by inhibitors of transcription, and it has been proposed that ATM responds to RNA⅐DNA hybrid R-loops that form at stalled RNA polymerase II transcription bubbles (7). However, the detection and signaling of CPT-induced, transcription-dependent DNA damage are not well understood, nor is it clear