ATR kinase regulates its attenuation via PPM1D phosphatase recruitment to chromatin during recovery from DNA replication stress signalling

In eukaryotes, in response to replication stress, DNA damage response kinase, ATR is activated, whose signalling abrogation leads to cell lethality due to aberrant fork remodelling and excessive origin firing. Here we report that inhibition of ATR kinase activity specifically during replication stress recovery results in persistent ATR signalling, evidenced by the presence of ATR-dependent phosphorylation marks (γH2AX, pChk1 and pRad17) and delayed cell cycle re-entry. Further, such disruption of ATR signalling attenuation leads to double-strand breaks, fork collapse and thereby ‘replication catastrophe’. PPM1D phosphatase, a nucleolar localized protein, relocates to chromatin during replication stress and reverts back to nucleolus following stress recovery, under the control of ATR kinase action. Inhibition of ATR kinase activity, specifically during post replication stress, triggers dislodging of the chromatin-bound PPM1D from nucleus to cytoplasm followed by its degradation, thereby leading to persistence of activated ATR marks in the nuclei. Chemical inhibition of PPM1D activity or SiRNA mediated depletion of the protein during post replication stress recovery ‘phenocopies’ ATR kinase inhibition by failing to attenuate ATR signalling. Collectively, our observations suggest a novel role of ATR kinase in mediating its own signal attenuation via PPM1D recruitment to chromatin as an essential mechanism for restarting the stalled forks, cell-cycle re-entry and cellular recovery from replication stress.     

TOPBP1 regulates RAD51 phosphorylation and chromatin loading and determines PARP inhibitor sensitivity

Topoisomerase IIβ-binding protein 1 (TOPBP1) participates in DNA replication and DNA damage response; however, its role in DNA repair and relevance for human cancer remain unclear. Here, through an unbiased small interfering RNA screen, we identified and validated TOPBP1 as a novel determinant whose loss sensitized human cells to olaparib, an inhibitor of poly(ADP-ribose) polymerase. We show that TOPBP1 acts in homologous recombination (HR) repair, impacts olaparib response, and exhibits aberrant patterns in subsets of human ovarian carcinomas. TOPBP1 depletion abrogated RAD51 loading to chromatin and formation of RAD51 foci, but without affecting the upstream HR steps of DNA end resection and RPA loading. Furthermore, TOPBP1 BRCT domains 7/8 are essential for RAD51 foci formation. Mechanistically, TOPBP1 physically binds PLK1 and promotes PLK1 kinase–mediated phosphorylation of RAD51 at serine 14, a modification required for RAD51 recruitment to chromatin. Overall, our results provide mechanistic insights into TOPBP1’s role in HR, with potential clinical implications for cancer treatment.     

Henipaviruses and lyssaviruses target nucleolar treacle protein and regulate ribosomal RNA synthesis

The nucleolus is a common target of viruses and viral proteins, but for many viruses the functional outcomes and significance of this targeting remains unresolved. Recently, the first intranucleolar function of a protein of a cytoplasmically-replicating negative-sense RNA virus (NSV) was identified, with the finding that the matrix (M) protein of Hendra virus (HeV) (genus Henipavirus, family Paramyxoviridae) interacts with Treacle protein within nucleolar subcompartments and mimics a cellular mechanism of the nucleolar DNA-damage response (DDR) to suppress ribosomal RNA (rRNA) synthesis. Whether other viruses utilise this mechanism has not been examined. We report that sub-nucleolar Treacle targeting and modulation is conserved between M proteins of multiple Henipaviruses, including Nipah virus and other potentially zoonotic viruses. Furthermore, this function is also evident for P3 protein of rabies virus, the prototype virus of a different RNA virus family (Rhabdoviridae), with Treacle depletion in cells also found to impact virus production. These data indicate that unrelated proteins of viruses from different families have independently developed nucleolar/Treacle targeting function, but that modulation of Treacle has distinct effects on infection. Thus, subversion of Treacle may be an important process in infection by diverse NSVs, and so could provide novel targets for antiviral approaches with broad specificity.     

Tim–Tipin dysfunction creates an indispensible reliance on the ATR–Chk1 pathway for continued DNA synthesis

The Tim (Timeless)–Tipin complex has been proposed to maintain genome stability by facilitating ATR-mediated Chk1 activation. However, as a replisome component, Tim–Tipin has also been suggested to couple DNA unwinding to synthesis, an activity expected to suppress single-stranded DNA (ssDNA) accumulation and limit ATR–Chk1 pathway engagement. We now demonstrate that Tim–Tipin depletion is sufficient to increase ssDNA accumulation at replication forks and stimulate ATR activity during otherwise unperturbed DNA replication. Notably, suppression of the ATR–Chk1 pathway in Tim–Tipin-deficient cells completely abrogates nucleotide incorporation in S phase, indicating that the ATR-dependent response to Tim–Tipin depletion is indispensible for continued DNA synthesis. Replication failure in ATR/Tim-deficient cells is strongly associated with synergistic increases in H2AX phosphorylation and DNA double-strand breaks, suggesting that ATR pathway activation preserves fork stability in instances of Tim–Tipin dysfunction. Together, these experiments indicate that the Tim–Tipin complex stabilizes replication forks both by preventing the accumulation of ssDNA upstream of ATR–Chk1 function and by facilitating phosphorylation of Chk1 by ATR.     

DNA-activated protein kinase functions in a newly observed S phase checkpoint that links histone mRNA abundance with DNA replication

DNA and histone synthesis are coupled and ongoing replication is required to maintain histone gene expression. Here, we expose S phase–arrested cells to the kinase inhibitors caffeine and LY294002. This uncouples DNA replication from histone messenger RNA (mRNA) abundance, altering the efficiency of replication stress–induced histone mRNA down-regulation. Interference with caffeine-sensitive checkpoint kinases ataxia telangiectasia and Rad3 related (ATR)/ataxia telangiectasia mutated (ATM) does not affect histone mRNA down- regulation, which indicates that ATR/ATM alone cannot account for such coupling. LY294002 potentiates caffeine's ability to uncouple histone mRNA stabilization from replication only in cells containing functional DNA-activated protein kinase (DNA-PK), which indicates that DNA-PK is the target of LY294002. DNA-PK is activated during replication stress and DNA-PK signaling is enhanced when ATR/ATM signaling is abrogated. Histone mRNA decay does not require Chk1/Chk2. Replication stress induces phosphorylation of UPF1 but not hairpin-binding protein/stem-loop binding protein at S/TQ sites, which are preferred substrate recognition motifs of phosphatidylinositol 3-kinase–like kinases, which indicates that histone mRNA stability may be directly controlled by ATR/ATM- and DNA-PK–mediated phosphorylation of UPF1.     

Replication stress by Py–Im polyamides induces a non-canonical ATR-dependent checkpoint response

Pyrrole–imidazole polyamides targeted to the androgen response element were cytotoxic in multiple cell lines, independent of intact androgen receptor signaling. Polyamide treatment induced accumulation of S-phase cells and of PCNA replication/repair foci. Activation of a cell cycle checkpoint response was evidenced by autophosphorylation of ATR, the S-phase checkpoint kinase, and by recruitment of ATR and the ATR activators RPA, 9-1-1, and Rad17 to chromatin. Surprisingly, ATR activation was accompanied by only a slight increase in single-stranded DNA, and the ATR targets RPA2 and Chk1, a cell cycle checkpoint kinase, were not phosphorylated. However, ATR activation resulted in phosphorylation of the replicative helicase subunit MCM2, an ATR effector. Polyamide treatment also induced accumulation of monoubiquitinated FANCD2, which is recruited to stalled replication forks and interacts transiently with phospho-MCM2. This suggests that polyamides induce replication stress that ATR can counteract independently of Chk1 and that the FA/BRCA pathway may also be involved in the response to polyamides. In biochemical assays, polyamides inhibit DNA helicases, providing a plausible mechanism for S-phase inhibition.     

Loss of Peter Pan protein is associated with cell cycle defects and apoptotic events

The nucleolus is a subnuclear compartment, which governs ribosome biogenesis. Moreover, it functions as hub in the stress response by orchestrating a variety of processes, such as regulation of cell cycle progression, senescence and apoptosis. Emerging evidence links the nucleolus also to the control of genomic stability and the development of human malignancies. Peter Pan (PPAN) is an essential ribosome biogenesis factor localized to nucleoli and mitochondria. We earlier showed that PPAN depletion triggers p53-independent nucleolar stress and apoptosis. In this study we investigated the precise localization of nucleolar PPAN during cell cycle and its function in cell cycle regulation. We show that PPAN knockdown impairs cell proliferation and induces G0/G1 as well as later G2/M cell cycle arrest in cancer cells. Although PPAN knockdown stabilizes the tumor suppressor p53 and induces CDKN1A/p21, the proliferation defects occur largely in a p53/p21-independent manner. We noticed a reduced number of knockdown cells entering cytokinesis and an elevation of binucleation. PPAN knockdown is also associated with increased H2A.X phosphorylation (γH2A.X) in cancer cells. We evaluated a potential signaling axis through the DNA damage response kinases ATM and ATR and alternatively apoptosis as a potent driver of γH2A.X. Interestingly, PPAN knockdown does not involve activation of ATM/ATR. Instead, γH2A.X is generated as a consequence of apoptosis induction in cancer cells. Strikingly, PPAN depletion in human fibroblasts did neither provoke apoptosis nor H2A.X phosphorylation, but recapitulated p53 stabilization. In summary, our data underline the notion that the PPAN-mediated, p53-independent nucleolar stress response has multiple facets.     

TopBP1 and DNA polymerase-α directly recruit the 9-1-1 complex to stalled DNA replication forks

TopBP1 and the Rad9–Rad1–Hus1 (9-1-1) complex activate the ataxia telangiectasia mutated and Rad3-related (ATR) protein kinase at stalled replication forks. ATR is recruited to stalled forks through its binding partner, ATR-interacting protein (ATRIP); however, it is unclear how TopBP1 and 9-1-1 are recruited so that they may join ATR–ATRIP and initiate signaling. In this study, we use Xenopus laevis egg extracts to determine the requirements for 9-1-1 loading. We show that TopBP1 is required for the recruitment of both 9-1-1 and DNA polymerase (pol)-α to sites of replication stress. Furthermore, we show that pol-α is also directly required for Rad9 loading. Our study identifies an assembly pathway, which is controlled by TopBP1 and includes pol-α, that mediates the loading of the 9-1-1 complex onto stalled replication forks. These findings clarify early events in the assembly of checkpoint signaling complexes on DNA and identify TopBP1 as a critical sensor of replication stress.     

Increased Stability of Nucleolar PinX1 in the Presence of TERT

PinX1, a nucleolar protein of 328 amino acids, inhibits telomerase activity, which leads to the shortening of telomeres. The C-terminal region of PinX1 is responsible for its nucleolar localization and binding with TERT, a catalytic component of telomerase. A fraction of TERT localizes to the nucleolus, but the role of TERT in the nucleolus is largely unknown. Here, we report a functional connection between PinX1 and TERT regarding PinX1 stability. The C-terminal of PinX1^205–328, a nucleolar fragment, was much more stable than the N-terminal of PinX1^1–204, a nuclear fragment. Interestingly, PinX1 was less stable in TERT-depleted cells and more stable in TERT-myc expressing cells. Stability assays for PinX1 truncation forms showed that both PinX1^1–328 and PinX1^205–328, nucleolar forms, were more rapidly degraded in TERT-depleted cells, while they were more stably maintained in TERT-overexpressing cells, compared to each of the controls. However, PinX1^1–204 was degraded regardless of the TERT status. These results reveal that the stability of PinX1 is maintained in nucleolus in the presence of TERT and suggest a role of TERT in the regulation of PinX1 steady-state levels.     

TOPBP1 takes RADical command in recombinational DNA repair

TOPBP1 is a key player in DNA replication and DNA damage signaling. In this issue, Moudry et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201507042) uncover a crucial role for TOPBP1 in DNA repair by revealing its requirement for RAD51 loading during repair of double strand breaks by homologous recombination.Proper replication and maintenance of the eukaryotic genome requires the involvement of the scaffolding protein TOPBP1. Over the last 20 years, studies in yeast, frog, and mammals have revealed conserved roles for TOPBP1 in initiation of DNA replication and activation of DNA damage signaling. TOPBP1 has been shown to assemble ternary protein complexes necessary to jump-start DNA replication or to initiate DNA damage signaling events by recognizing distinct phosphoproteins via its multiple BRCA1 C terminus (BRCT) domains (Fig. 1 D; Wardlaw et al., 2014). In this issue, Moudry et al. add to the list of crucial TOPBP1 roles in genome biology and reveal that TOPBP1 is also required for proper repair of double strand breaks (DSBs) by homologous recombination (HR).Open in a separate windowFigure 1.Involvement of TOPBP1 in HR-mediated repair. (A) The work by Moudry et al. (2016) supports a model in which TOPBP1 directs the action of PLK1 kinase toward RAD51, mediating a phosphorylation event that licenses a second phosphorylation event mediated by the casein kinase 2 (CK2) kinase. These phosphorylation events are believed to facilitate the loading of RAD51, which replaces RPA, at damage sites and favor HR. (B) TOPBP1 as a hub for coordinating the action of multiple kinases toward genome maintenance. (C) Speculative model for the mutually exclusive engagement of TOPBP1 in distinct cellular processes as a strategy for coordinating genome replication and DNA damage responses (see text for details). (D) Current understanding of how TOPBP1 and its yeast orthologue Dpb11 mediate the formation of ternary complexes for replication initiation, DNA damage signaling, and recombinational repair. In mammals, the indicated proteins have been shown to interact with TOPBP1, but the roles of most of those interactions remain unclear. For simplicity, some TOPBP1 interactions and their cofactors are not depicted.Moudry et al. (2016) report that depletion of TOPBP1 makes cells highly sensitive to the poly (ADP-ribose) polymerase inhibitor olaparib, a drug known to sensitize cells with an already dysfunctional HR machinery. In particular, olaparib hypersensitizes cells that carry mutations in the bona fide HR factors and tumor suppressors BRCA1 or BRCA2. In this work, the authors first identified TOPBP1 as a hit in a high-content RNAi screen for proteins whose depletion resulted in higher toxicity after olaparib treatment in osteosarcoma cells, which suggests that loss or inactivation of TOPBP1 predicts the response of cancer cells to this drug. Moudry et al. (2016) observed that RNAi-mediated knockdown of TOPBP1 in cancer cells treated with olaparib increased the level of DNA damage and induced DNA DSB markers. The researchers subsequently examined whether olaparib sensitivity reflected defective HR in TOPBP1-depleted cells by measuring HR activity through several parameters and confirmed that TOPBP1-depleted cells showed reduced HR activity.The HR process encompasses several phases, including end resection and chromatin loading of RPA and RAD51, which can be visualized by formation of microscopically detectable foci. Moudry et al. (2016) searched for which step of HR was compromised in cells depleted for TOPBP1 and found that DNA end resection, i.e., the processing of the 5′ recessed end that exposes a 3′ overhang used for homology search, seemed not to be affected, as evaluated by the amounts of single stranded DNA detected by BrdU incorporation under nondenaturing conditions. Interestingly, they found that the next key stage in HR, in which the RAD51 recombinase protein is loaded at these 3′ overhangs (Fig. 1 A), was greatly impaired, based on the assessment of the formation of RAD51 foci by microscopy and of the biochemical analysis of RAD51 accumulation on chromatin. Although the mechanism by which TOPBP1 promotes the loading of RAD51 remains unclear, the authors propose an interesting model in which TOPBP1 plays a scaffolding role to direct Polo-like Kinase 1 (PLK1), which phosphorylates RAD51 and facilitates its loading to DNA damage sites (Fig. 1 A; Yata et al., 2012). Consistent with this model, they show that TOPBP1 physically interacts with PLK1 and that depletion of TOPBP1 impairs PLK1-dependent RAD51 phosphorylation. Although more work is needed to prove that the TOPBP1–PLK1 interaction is required for this phosphorylation event, the results are exciting as they suggest another important functional link between TOPBP1 and a kinase. During DNA damage signaling, TOPBP1 plays an established role in activating the ATR kinase (Kumagai et al., 2006) and is believed to direct ATR’s action toward specific substrates. This latter function is best understood in yeast, in which TOPBP1/Dpb11 forms a ternary complex to direct ATR/Mec1 action to phosphorylate the downstream kinase Rad53. Interestingly, recent data from fission yeast also suggest that TOPBP1 interacts with yet another kinase, CDK, and directs its kinase action (Qu et al., 2013). The emerging scenario is that TOPBP1 may function as a scaffolding hub for controlling the action of distinct kinases to ensure genome integrity (Fig. 1 B).Although the work of Moudry et al. (2016) is the first to show a clear role for TOPBP1 in RAD51 loading, studies in budding yeast have proposed links between the TOPBP1 orthologue Dpb11 and HR-mediated repair. It was shown that the temperature-sensitive dpb11-1 mutant displays a sensitivity to DNA damage that is not further increased by deletion of RAD51, suggesting that Dpb11 functions in HR repair (Ogiwara et al., 2006). In addition, other groups showed that TOPBP1/Dpb11 is required for DSB-induced mating-type switching and also reached the conclusion that TOPBP1/Dpb11 is required for HR-mediated repair of a DSB (Germann et al., 2011; Hicks et al., 2011). These studies provided compelling evidence that the role for TOPBP1/Dpb11 in DSB repair is independent of its roles in replication initiation and DNA damage signaling. In humans, there also is evidence pointing to potential roles for TOPBP1 in DNA repair, as depletion of TOPBP1 was found to increase sensitivity to ionizing radiation and lead to defective DSB repair by HR (Morishima et al., 2007).The new set of results provided by Moudry et al. (2016) clearly place TOPBP1 at the center stage of HR-mediated repair in what seems to be yet another key and evolutionarily conserved role for TOPBP1, in addition to replication initiation and DNA damage signaling. An intriguing and unanswered question relates to defining the evolutionary benefit conferred by maintaining these crucial roles in the same protein. It is tempting to speculate that having a single protein module in command of key licensing events helps ensure the ordered and mutually exclusive execution of distinct cellular processes (Fig. 1 C). This is a particularly attractive and well-suited idea for the established role of DNA damage signaling in inhibiting origin firing during DNA replication. Sequestration of TOPBP1 into a complex involved in DNA damage signaling would help ensure that replication initiation is inhibited. Consistent with this hypothesis, it is established in yeast that the same BRCT domains involved in replication initiation are also required for DNA damage signaling. In addition, it was recently shown that competition between DNA damage signaling proteins and DNA repair factors for binding to the BRCT domains of TOPBP1/Dpb11 is a mechanism to remove TOPBP1/Dpb11 from a pro-DNA damage signaling complex, resulting in dampening of DNA damage signaling (Ohouo et al., 2013; Cussiol et al., 2015). It will be exciting to further explore this competition-based regulatory mechanism in human cells, as well as in the coordination of DNA damage signaling with DNA repair. In this direction, it is crucial that the precise molecular mechanism by which TOPBP1 promotes HR repair is elucidated, including defining which TOPBP1 BRCT domains are required and which factors they are binding to favor RAD51 loading or other pro-HR functions. Through truncation mutation analyses, Moudry et al. (2016) show that the specific BRCT domains 7/8 of TOPBP1 are essential for TOPBP1’s role in promoting HR. However, it remains unclear how this is accomplished mechanistically.To make the scenario even more complicated, TOPBP1 is known to physically interact with an extensive network of repair factors, including, but not limited to, BRCA1, 53BP1, MRN, FANCJ, and BLM (Greenberg et al., 2006; Wardlaw et al., 2014). This points to an extremely complex system by which TOPBP1 could be coordinating the action of a range of repair factors and repair pathways (Fig. 1 D). It would not be surprising if TOPBP1 was found to be key for the regulation of other steps in HR-mediated repair as well as other repair pathways in response to varied types of genotoxic insults, including DNA replication stress. In yeast, the interaction between TOPBP1/Dpb11 and the repair scaffold Slx4 provides an additional example of the rich range of possibilities by which TOPBP1/Dpb11 functions in DNA repair. In addition to sequestering TOPBP1/Dpb11 and dampening DNA damage signaling (Ohouo et al., 2013; Cussiol et al., 2015), the Slx4–TOPBP1/Dpb11 interaction was recently found to control DNA end resection (Dibitetto et al., 2015) and was proposed to affect the late step of resolution of repair intermediates (Gritenaite et al., 2014). The TOPBP1–SLX4 interaction is conserved in humans; however, it remains unclear how this interaction impacts DNA repair in higher eukaryotes. Moreover, whereas in yeast it is possible to clearly define a pro-DNA damage signaling complex and a pro-recombinational repair complex (Fig. 1 D), in mammals the scenario is more complex and it is currently unclear what the precise contributions of different TOPBP1 interactions are in DNA damage signaling and/or recombinational repair. Finally, because the ATR kinase is expected to regulate several DNA repair factors, it is likely that the ATR-activating function of TOPBP1 plays important roles in some aspect of DNA repair. A major experimental avenue to explore this possibility and improve our understanding of the other roles for TOPBP1 in DNA repair will be the generation of separation-of-function mutants that do not interfere with DNA replication or DNA damage signaling.Following the findings reported by Moudry et al. (2016), it is interesting to speculate on the implications of understanding TOPBP1’s role in HR-mediated repair for cancer research and treatment. Little is known about the role of TOPBP1 in carcinogenesis. It was found that TOPBP1 expression and subcellular localization are altered in a subset of breast cancer samples (Going et al., 2007; Liu et al., 2009; Forma et al., 2012) and Moudry et al. (2016) also report altered TOPBP1 protein expression in ovarian cancers, although at modest frequencies. Nonetheless, as we learn more about TOPBP1 mechanisms of action in HR, it is possible that it may become an important target for manipulating the HR response by using small molecules such as Calcein AM, which targets BRCT domains 7/8 of TOPBP1 (Chowdhury et al., 2014) and was shown by Moudry et al. (2016) to impair HR. Concerning the finding that TOPBP1 plays a pro-HR function very much like BRCA1 and BRCA2, whose genes are most frequently mutated in ovarian and breast cancers, it is intriguing that although TOPBP1 mutations have been found in cancers (Rebbeck et al., 2009; Forma et al., 2013), they are relatively infrequent and are likely not driver mutations. If TOPBP1 plays a key role in RAD51 loading, which is the step severely perturbed in BRCA1- or BRCA2-mutated cancer cells, it is not clear how more cancer-driving mutations have not been identified in TOPBP1. One possibility is that TOPBP1 mutations affecting TOPBP1’s pro-HR function also affect DNA replication and DNA damage signaling and impair the replicative capacity of cancer cells. Disentangling potential antagonistic roles for TOPBP1 in both suppressing and supporting tumorigenesis could lead to exciting new directions to study this complex multifunctional protein and to potentially develop new therapeutic strategies.     

DNA structure-specific priming of ATR activation by DNA-PKcs

Three phosphatidylinositol-3-kinase–related protein kinases implement cellular responses to DNA damage. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia-telangiectasia mutated respond primarily to DNA double-strand breaks (DSBs). Ataxia-telangiectasia and RAD3-related (ATR) signals the accumulation of replication protein A (RPA)–covered single-stranded DNA (ssDNA), which is caused by replication obstacles. Stalled replication intermediates can further degenerate and yield replication-associated DSBs. In this paper, we show that the juxtaposition of a double-stranded DNA end and a short ssDNA gap triggered robust activation of endogenous ATR and Chk1 in human cell-free extracts. This DNA damage signal depended on DNA-PKcs and ATR, which congregated onto gapped linear duplex DNA. DNA-PKcs primed ATR/Chk1 activation through DNA structure-specific phosphorylation of RPA32 and TopBP1. The synergistic activation of DNA-PKcs and ATR suggests that the two kinases combine to mount a prompt and specific response to replication-born DSBs.     

Mutations in Replicative Stress Response Pathways Are Associated with S Phase-specific Defects in Nucleotide Excision Repair

Nucleotide excision repair (NER) is a highly conserved pathway that removes helix-distorting DNA lesions induced by a plethora of mutagens, including UV light. Our laboratory previously demonstrated that human cells deficient in either ATM and Rad3-related (ATR) kinase or translesion DNA polymerase η (i.e. key proteins that promote the completion of DNA replication in response to UV-induced replicative stress) are characterized by profound inhibition of NER exclusively during S phase. Toward elucidating the mechanistic basis of this phenomenon, we developed a novel assay to quantify NER kinetics as a function of cell cycle in the model organism Saccharomyces cerevisiae. Using this assay, we demonstrate that in yeast, deficiency of the ATR homologue Mec1 or of any among several other proteins involved in the cellular response to replicative stress significantly abrogates NER uniquely during S phase. Moreover, initiation of DNA replication is required for manifestation of this defect, and S phase NER proficiency is correlated with the capacity of individual mutants to respond to replicative stress. Importantly, we demonstrate that partial depletion of Rfa1 recapitulates defective S phase-specific NER in wild type yeast; moreover, ectopic RPA1–3 overexpression rescues such deficiency in either ATR- or polymerase η-deficient human cells. Our results strongly suggest that reduction of NER capacity during periods of enhanced replicative stress, ostensibly caused by inordinate sequestration of RPA at stalled DNA replication forks, represents a conserved feature of the multifaceted eukaryotic DNA damage response.     

SLX4–XPF mediates DNA damage responses to replication stress induced by DNA–protein interactions

The DNA damage response (DDR) has a critical role in the maintenance of genomic integrity during chromosome replication. However, responses to replication stress evoked by tight DNA–protein complexes have not been fully elucidated. Here, we used bacterial LacI protein binding to lacO arrays to make site-specific replication fork barriers on the human chromosome. These barriers induced the accumulation of single-stranded DNA (ssDNA) and various DDR proteins at the lacO site. SLX4–XPF functioned as an upstream factor for the accumulation of DDR proteins, and consequently, ATR and FANCD2 were interdependently recruited. Moreover, LacI binding in S phase caused underreplication and abnormal mitotic segregation of the lacO arrays. Finally, we show that the SLX4–ATR axis represses the anaphase abnormality induced by LacI binding. Our results outline a long-term process by which human cells manage nucleoprotein obstacles ahead of the replication fork to prevent chromosomal instability.     

NOA36 Protein Contains a Highly Conserved Nucleolar Localization Signal Capable of Directing Functional Proteins to the Nucleolus,in Mammalian Cells

NOA36/ZNF330 is an evolutionarily well-preserved protein present in the nucleolus and mitochondria of mammalian cells. We have previously reported that the pro-apoptotic activity of this protein is mediated by a characteristic cysteine-rich domain. We now demonstrate that the nucleolar localization of NOA36 is due to a highly-conserved nucleolar localization signal (NoLS) present in residues 1–33. This NoLS is a sequence containing three clusters of two or three basic amino acids. We fused the amino terminal of NOA36 to eGFP in order to characterize this putative NoLS. We show that a cluster of three lysine residues at positions 3 to 5 within this sequence is critical for the nucleolar localization. We also demonstrate that the sequence as found in human is capable of directing eGFP to the nucleolus in several mammal, fish and insect cells. Moreover, this NoLS is capable of specifically directing the cytosolic yeast enzyme polyphosphatase to the target of the nucleolus of HeLa cells, wherein its enzymatic activity was detected. This NoLS could therefore serve as a very useful tool as a nucleolar marker and for directing particular proteins to the nucleolus in distant animal species.