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The proper cellular response to DNA double-strand breaks (DSBs) is critical for maintaining the integrity of the genome. RecQL4, a DNA helicase of which mutations are associated with Rothmund-Thomson syndrome (RTS), is required for the DNA DSB response. However, the mechanism by which RecQL4 performs these essential roles in the DSB response remains unknown. Here, we show that RecQL4 and its helicase activity are required for maintaining the stability of the Mre11-Rad50-Nbs1 (MRN) complex on DSB sites during a DSB response. We found using immunocytochemistry and live-cell imaging that the MRN complex is prematurely disassembled from DSB sites in a manner dependent upon Skp2-mediated ubiquitination of Nbs1 in RecQL4-defective cells. This early disassembly of the MRN complex could be prevented by altering the ubiquitination site of Nbs1 or by expressing a deubiquitinase, Usp28, which sufficiently restored homologous recombination repair and ATM, a major checkpoint kinase against DNA DSBs, activation abilities in RTS, and RecQL4-depleted cells. These results suggest that the essential role of RecQL4 in the DSB response is to maintain the stability of the MRN complex on DSB sites and that defects in the DSB response in cells of patients with RTS can be recovered by controlling the stability of the MRN complex.

Sequential activation of DNA replication origins is precisely programmed and critical to maintaining genome stability. RecQL4, a member of the conserved RecQ family of helicases, plays an essential role in the initiation of DNA replication in mammalian cells. Here, we showed that RecQL4 protein tethered on the pre-replicative complex (pre-RC) induces early activation of late replicating origins during S phase. Tethering of RecQL4 or its N terminus on pre-RCs via fusion with Orc4 protein resulted in the recruitment of essential initiation factors, such as Mcm10, And-1, Cdc45, and GINS, increasing nascent DNA synthesis in late replicating origins during early S phase. In this origin activation process, tethered RecQL4 was able to recruit Cdc45 even in the absence of cyclin-dependent kinase (CDK) activity, whereas CDK phosphorylation of RecQL4 N terminus was required for interaction with and origin recruitment of And-1 and GINS. In addition, forced activation of replication origins by RecQL4 tethering resulted in increased replication stress and the accumulation of ssDNAs, which can be recovered by transcription inhibition. Collectively, these results suggest that recruitment of RecQL4 to replication origins is an important step for temporal activation of replication origins during S phase. Further, perturbation of replication timing control by unscheduled origin activation significantly induces replication stress, which is mostly caused by transcription-replication conflicts.

RecQL4 has been shown to be involved in DNA replication and repair, but its role in DNA damage checkpoint pathway has not been reported. Here, we show that RecQL4 plays an important role in the activation of ataxia telangiectasia mutated (ATM)-dependent checkpoint pathway in human cells. Cells depleted with RecQL4 or Rothmund-Thomson syndrome cells showed significant impairment in the activation of ATM and the downstream effector proteins such as checkpoint kinase 2 and p53 after DNA damage. This defect was recovered with the expression of wild type RecQL4 but not any mutant RecQL4 proteins with defective helicase activities. While RecQL4 failed to show any direct interaction with ATM, it stably interacted with the Mre11-Rad50-Nbs1 complex that is essential for the activation of ATM and was localized on the DNA damage foci. Thus, our results suggest that the helicase activity of RecQL4 plays an important role in the activation of ATM-dependent checkpoint pathway against DNA double strand breaks in human cells.

Though the G(1) checkpoint in mammalian cells has been known for decades, the molecular targets that prevent S-phase entry remain unknown. Mimosine is a rare plant amino acid that arrests the cell cycle in the G(1) phase before entry into S phase. Here, we show that mimosine interrupts the binding of Ctf4 to chromatin, which is essential for the initiation of DNA replication in HeLa cells, and this effect is mediated by the Hif-1α-dependent increase in the level of p27. Depletion of Hif-1α results in an increased binding of Ctf4 to chromatin and the entry of cells into S phase even in the presence of mimosine. These results suggest that the binding of Ctf4 to chromatin is the target of the Hif-1α-dependent checkpoint pathway for cell cycle arrest in G(1) phase. Although we observed Hif-1α-dependent arrest in mimosine-treated cells, it is possible that Ctf4 may act as a common target for G(1) arrest in various other checkpoint pathways.

In eukaryotes, the activation of the prereplicative complex and assembly of an active DNA unwinding complex are critical but poorly understood steps required for the initiation of DNA replication. In this report, we have used bimolecular fluorescence complementation assays in HeLa cells to examine the interactions between Cdc45, Mcm2–7, and the GINS complex (collectively called the CMG complex), which seem to play a key role in the formation and progression of replication forks. Interactions between the CMG components were observed only after the G 1 /S transition of the cell cycle and were abolished by treatment of cells with either a CDK inhibitor or siRNA against the Cdc7 kinase. Stable association of CMG required all three components of the CMG complex as well as RecQL4, Ctf4/And-1, and Mcm10. Surprisingly, depletion of TopBP1, a homologue of Dpb11 that plays an essential role in the chromatin loading of Cdc45 and GINS in yeast cells, did not significantly affect CMG complex formation. These results suggest that the proteins involved in the assembly of initiation complexes in human cells may differ somewhat from those in yeast systems.

The initiation of eukaryotic DNA replication requires assembly of the pre‐replicative complex (Pre‐RC) through the concerted action of Orc, Cdc6, Cdt1 and Mcm2–7 complex during G1 phase. The pre‐RC assembly licenses individual replication origins for the initiation of DNA replication and sufficient number of the pre‐RC is essential for proper progression of S phase. However, it is not well known how cells recognize the completion of the pre‐RC assembly before G1‐S transition. In order to understand the cellular responses to the defects in pre‐RC assembly, we depleted the known components of pre‐RC proteins using the small interference RNAs in HeLa cells. Although the defects of pre‐RC assembly by the depletion of the pre‐RC proteins such as Orc2, Cdt1, Mcm2 & Mcm10 did not elicit the activation of Chk1‐ or Chk2‐dependent checkpoint pathways, these cells still showed significant decrease in the cellular level of Cdc25A proteins. These results suggests that a novel checkpoint pathway exist in HeLa ...

MCM8-9 complex is required for homologous recombination (HR)-mediated repair of double-strand breaks (DSBs). Here we report that MCM8-9 is required for DNA resection by MRN (MRE11-RAD50-NBS1) at DSBs to generate ssDNA. MCM8-9 interacts with MRN and is required for the nuclease activity and stable association of MRN with DSBs. The ATPase motifs of MCM8-9 are required for recruitment of MRE11 to foci of DNA damage. Homozygous deletion of the MCM9 found in various cancers sensitizes a cancer cell line to interstrand-crosslinking (ICL) agents. A cancer-derived point mutation or an SNP on MCM8 associated with premature ovarian failure (POF) diminishes the functional activity of MCM8. Therefore, the MCM8-9 complex facilitates DNA resection by the MRN complex during HR repair, genetic or epigenetic inactivation of MCM8 or MCM9 are seen in human cancers, and genetic inactivation of MCM8 may be the basis of a POF syndrome., The MCM8-9 complex is required for repair of DNA double-strand breaks (DSBs). Here, the authors show that MCM8-9 is required for the nuclease activity of the MRE11-RAD50-NBS1 complex (MRN) and stabilizes its association with DNA damage sites, promoting resection of DSB ends.

Though RecQL4 was shown to be essential for the initiation of DNA replication in mammalian cells, its role in initiation is poorly understood. Here, we show that RecQL4 is required for the origin binding of Mcm10 and Ctf4, and their physical interactions and association with replication origins are controlled by the concerted action of both CDK and DDK activities. Although RecQL4-dependent binding of Mcm10 and Ctf4 to chromatin can occur in the absence of pre-replicative complex, their association with replication origins requires the presence of the pre-replicative complex and CDK and DDK activities. Their association with replication origins and physical interactions are also targets of the DNA damage checkpoint pathways which prevent initiation of DNA replication at replication origins. Taken together, the RecQL4-dependent association of Mcm10 and Ctf4 with replication origins appears to be the first important step controlled by S phase promoting kinases and checkpoint pathways for the initiation of DNA replication in human cells.

Summary Double-strand breaks (DSBs) of DNA in eukaryotic cells are predominantly repaired by non-homologous end joining (NHEJ). The histone chaperone anti-silencing factor 1a (ASF1a) interacts with MDC1 and is recruited to sites of DSBs to facilitate the interaction of phospho-ATM with MDC1 and phosphorylation of MDC1, which are required for the recruitment of RNF8/RNF168 histone ubiquitin ligases. Thus, ASF1a deficiency reduces histone ubiquitination at DSBs, decreasing the recruitment of 53BP1, and decreases NHEJ, rendering cells more sensitive to DSBs. This role of ASF1a in DSB repair cannot be provided by the closely related ASF1b and does not require its histone chaperone activity. Homozygous deletion of ASF1A is seen in 10%–15% of certain cancers, suggesting that loss of NHEJ may be selected in some malignancies and that the deletion can be used as a molecular biomarker for cancers susceptible to radiotherapy or to DSB-inducing chemotherapy.

Chromosomal DNA replication is a highly conserved process in all eukaryotes that starts at specific sites along DNA known as replication origins. The activation of DNA replication origins requires assembly of prereplicative complexes (pre-RCs) followed by recruitment of additional initiating factors and formation of replisomes (Bell and Dutta 2002; Sclafani and Holzen 2007). Assembly of the pre-RCs onto each replication origin occurs during the early G1 phase of the cell cycle through sequential recruitment of the origin recognition complex (ORC), CDC6, CDT1, and minichromosome maintenance 2–7 (MCM2–7 complex) proteins. However, excess MCM2–7 complexes are loaded, leading to the licensing of excess origins, most of which are dormant and passively replicated in the normal S phase. The excess licensed origins, however, serve as backup origins that can complete DNA replication when existing forks stall (Ibarra et al. 2008; Ge and Blow 2010). In the presence of DNA-damaging agents, when newly fired replication forks stall after replicating a few hundred bases, many additional (formerly dormant) origins begin firing in mammalian cells (Ge et al. 2007; Courbet et al. 2008; Karnani and Dutta 2011). DNA replication is often the target of anti-cancer drugs, leading to the generation of stalled replication forks (Hoeijmakers 2001). ATR kinase recognizes these stalled replication forks and activates the intra-S-phase checkpoint signaling cascade. The activation of ATR requires the replication protein A (RPA) complex RPA70–RPA32–RPA14 and ATRIP (Cortez et al. 2001; Ball et al. 2005; Cimprich and Cortez 2008). The RPA complex coats ssDNA at stalled replication forks and recruits ATR and other checkpoint proteins (Zou and Elledge 2003). Once activated, ATR triggers the checkpoint by phosphorylating many downstream targets (including RPA, CHK1, and p53), ultimately leading to arrest of the cell cycle in S phase. Since transcription of neighboring genes is often repressed by even a single DNA double-strand break (DSB) in mammalian cells by active pathways using ATM and DNA-dependent protein kinases (Kruhlak et al. 2007; Shanbhag et al. 2010; Pankotai et al. 2012), we wondered whether and how stalled replication forks produced by anti-cancer drugs affect the transcription of neighboring genes. Doxorubicin (DOX) is a widely used cancer chemotherapeutic drug that works by intercalating into dsDNA, inhibiting the activity of DNA topoisomerase II (Bodley et al. 1989; Capranico et al. 1990). Despite the extensive use and study of this drug, the locations of replication origins that fire in the presence of DOX, the locations of stalled forks, and the effects of the stalled forks on neighboring gene expression have not been studied. Previously, we found that hydroxyurea (HU), a ribonucleotide reductase inhibitor (Elford 1968), activates clusters of dormant replication origins that produce clusters of forks that stall after a few hundred bases of DNA synthesis (Karnani and Dutta 2011). Many of these neoreplication origin clusters overlap with coding genes. We therefore hypothesized that chemotherapy drugs like DOX could activate similar clusters of neoreplication origins and produce clusters of stalled replication forks so that the cis effects of these stalled forks on the local transcription machinery would be part of the gene expression changes seen in cells treated with DOX. We show that DOX treatment produces clusters of stalled replication forks at specific sites in the genome and that the production of those clusters decreases the transcription of neighboring genes due to active changes in the chromatin and detachment of RNA polymerase II. Surprisingly, the transcriptional repression was not simply due to the mechanical effects of local clusters of stalled forks but an ATR-dependent checkpoint pathway that down-regulates ASF1a, a histone chaperone known to act in association with replication forks. The CRL1βTRCPE3 ligase complex polyubiquitinates ASF1a and targets it for degradation by proteasomes. This is a new mechanism by which a globally active checkpoint pathway interacts with local clusters of stalled forks to specifically repress genes in the vicinity of the stalled forks. These specific changes in gene expression produced by anti-S-phase chemotherapy drugs like DOX will contribute to the efficacy or toxicity of this class of drugs. Finally, decrease of ASF1a makes cancer cells more sensitive to DOX, suggesting that the homozygous deletion or underexpression of this histone chaperone, as seen in several cancers, could be a personalized tumor marker for sensitivity to DOX.

Cdc7 is a serine/threonine kinase that plays essential roles in the initiation of eukaryotic DNA replication and checkpoint response. In previous studies, depletion of Cdc7 by small interfering RNA was shown to induce an abortive S phase that led to the cell cycle arrest in normal human fibroblasts and apoptotic cell death in various cancer cells. Here we report that stress-activated p38 MAP kinase was activated and responsible for apoptotic cell death in Cdc7-depleted HeLa cells. The activation of p38 MAP kinase in the Cdc7-depleted cells was shown to depend on ATR, a major sensor kinase for checkpoint or DNA damage responses. Only the p38 MAP kinase, and not the other stress-activated kinases such as JNK or ERK, was activated, and both caspase 8 and caspase 9 were activated for the induction of apoptosis. Activation of apoptosis in Cdc7-depleted cells was completely abolished in cells treated with small interfering RNA or an inhibitor of the p38 MAP kinase, suggesting that p38 MAP kinase activation was responsible for apoptotic cell death. Taken together, we suggest that the ATR-dependent activation of the p38 MAP kinase is a major signaling pathway that induces apoptotic cell death after depletion of Cdc7 in cancer cells.

Replicative senescence limits the proliferation of somatic cells passaged in culture and may reflect cellular aging in vivo. The most widely used biomarker for senescent and aging cells is senescence-associated beta-galactosidase (SA-beta-gal), which is defined as beta-galactosidase activity detectable at pH 6.0 in senescent cells, but the origin of SA-beta-gal and its cellular roles in senescence are not known. We demonstrate here that SA-beta-gal activity is expressed from GLB1, the gene encoding lysosomal beta-D-galactosidase, the activity of which is typically measured at acidic pH 4.5. Fibroblasts from patients with autosomal recessive G(M1)-gangliosidosis, which have defective lysosomal beta-galactosidase, did not express SA-beta-gal at late passages even though they underwent replicative senescence. In addition, late passage normal fibroblasts expressing small-hairpin interfering RNA that depleted GLB1 mRNA underwent senescence but failed to express SA-beta-gal. GLB1 mRNA depletion also prevented expression of SA-beta-gal activity in HeLa cervical carcinoma cells induced to enter a senescent state by repression of their endogenous human papillomavirus E7 oncogene. SA-beta-gal induction during senescence was due at least in part to increased expression of the lysosomal beta-galactosidase protein. These results also indicate that SA-beta-gal is not required for senescence.

The successful duplication of genomic DNA during S phase is essential for the proper transmission of genetic information to the next generation of cells. Perturbation of normal DNA replication by extrinsic stimuli or intrinsic stress can result in stalled replication forks, ultimately leading to abnormal chromatin structures and activation of the DNA damage response. On formation of stalled replication forks, many DNA repair and recombination pathway proteins are recruited to resolve the stalled fork and resume proper DNA synthesis. Initiation of replication at sites of stalled forks differs from traditional replication and, therefore, requires specialized proteins to reactivate DNA synthesis. In this issue of EMBO reports , Wan et al [1] introduce human primase‐polymerase 1 (hPrimpol1)/CCDC111, a novel factor that is essential for the restart of stalled replication forks. This article is the first, to our knowledge, to ascertain the function of human Primpol enzymes, which were originally identified as members of the archaeao‐eukaryotic primase (AEP) family [2]. Single‐stranded DNA (ssDNA) forms at stalled replication forks because of uncoupling of the DNA helicase from the polymerase, and is coated by replication protein A (RPA) for stabilization and recruitment of proteins involved in DNA repair and restart of replication. To identify novel factors playing important roles in the resolution of stalled replication forks, Wan and colleagues [1] used mass spectrometry to identify RPA‐binding partners. Among the proteins identified were those already known to be located at replication forks, including SMARCAL1/HARP, BLM and TIMELESS. In addition they found a novel interactor, the 560aa protein CCDC111. This protein interacts with the carboxyl terminus of RPA1 through its own C‐terminal region, and localizes with RPA foci in cells after hydroxyurea or DNA damage induced by ionizing irradiation. Owing to …

The E6 and E7 oncoproteins of human papillomavirus (HPV) play a major role in the development of cervical carcinoma. In this study, a recombinant adenovirus that expresses the bovine papillomavirus (BPV) E2, which has been shown to inhibit HPV early gene expression, was delivered to two HPV-immortalized cell lines as well as CaSki, a cervical carcinoma cell line. We tested whether the carcinoma and the immortal cells were equally affected by the expression of BPV E2. In all cell lines, BPV E2-mediated inhibition of HPV E6/E7 expression caused a dramatic suppression of cell growth, being preceded by the activation of the p53–Rb growth-inhibitory pathway, and a decrease in hTERT mRNA expression and telomerase activity. This suggests that the HPV E6 and E7 proteins are required not only for induction of the proliferative phenotype and telomerase activity, but also for their maintenance. In both the carcinoma and the immortal lines, the number of cells with enlarged cytoplasm and senescence-associated β-galactosidase activity, which are markers for cellular senescence, was significantly increased. These results suggest that a senescence program exists in cells immortalized by HPV DNA as well as in cervical carcinoma cells.