Tipin/Tim1/And1 protein complex promotes Polα chromatin binding and sister chromatid cohesion

The Tipin/Tim1 complex plays an important role in the S‐phase checkpoint and replication fork stability. However, the biochemical function of this complex is poorly understood. Using Xenopus laevis egg extract we show that Tipin is required for DNA replication in the presence of limiting amount of replication origins. Under these conditions the DNA replication defect correlates with decreased levels of DNA Polα on chromatin. We identified And1, a Polα chromatin‐loading factor, as new Tipin‐binding partner. We found that both Tipin and And1 promote stable binding of Polα to chromatin and that this is required for DNA replication under unchallenged conditions. Strikingly, extracts lacking Tipin and And1 also show reduced sister chromatids cohesion. These data indicate that Tipin/Tim1/And1 form a complex that links stabilization of replication fork and establishment of sister chromatid cohesion.     

Plx1 is required for chromosomal DNA replication under stressful conditions

Polo-like kinase (Plk)1 is required for mitosis progression. However, although Plk1 is expressed throughout the cell cycle, its function during S-phase is unknown. Using Xenopus laevis egg extracts, we demonstrate that Plx1, the Xenopus orthologue of Plk1, is required for DNA replication in the presence of stalled replication forks induced by aphidicolin, etoposide or reduced levels of DNA-bound Mcm complexes. Plx1 binds to chromatin and suppresses the ATM/ATR-dependent intra-S-phase checkpoint that inhibits origin firing. This allows Cdc45 loading and derepression of DNA replication initiation. Checkpoint activation increases Plx1 binding to the Mcm complex through its Polo box domain. Plx1 recruitment to chromatin is independent of checkpoint mediators Tipin and Claspin. Instead, ATR-dependent phosphorylation of serine 92 of Mcm2 is required for the recruitment of Plx1 to chromatin and for the recovery of DNA replication under stress. Depletion of Plx1 leads to accumulation of chromosomal breakage that is prevented by the addition of recombinant Plx1. These data suggest that Plx1 promotes genome stability by regulating DNA replication under stressful conditions.     

The human Tim/Tipin complex coordinates an Intra-S checkpoint response to UV that slows replication fork displacement

UV-induced DNA damage stalls DNA replication forks and activates the intra-S checkpoint to inhibit replicon initiation. In response to stalled replication forks, ATR phosphorylates and activates the transducer kinase Chk1 through interactions with the mediator proteins TopBP1, Claspin, and Timeless (Tim). Murine Tim recently was shown to form a complex with Tim-interacting protein (Tipin), and a similar complex was shown to exist in human cells. Knockdown of Tipin using small interfering RNA reduced the expression of Tim and reversed the intra-S checkpoint response to UVC. Tipin interacted with replication protein A (RPA) and RPA-coated DNA, and RPA promoted the loading of Tipin onto RPA-free DNA. Immunofluorescence analysis of spread DNA fibers showed that treating HeLa cells with 2.5 J/m(2) UVC not only inhibited the initiation of new replicons but also reduced the rate of chain elongation at active replication forks. The depletion of Tim and Tipin reversed the UV-induced inhibition of replicon initiation but affected the rate of DNA synthesis at replication forks in different ways. In undamaged cells depleted of Tim, the apparent rate of replication fork progression was 52% of the control. In contrast, Tipin depletion had little or no effect on fork progression in unirradiated cells but significantly attenuated the UV-induced inhibition of DNA chain elongation. Together, these findings indicate that the Tim-Tipin complex mediates the UV-induced intra-S checkpoint, Tim is needed to maintain DNA replication fork movement in the absence of damage, Tipin interacts with RPA on DNA and, in UV-damaged cells, Tipin slows DNA chain elongation in active replicons.     

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.     

Histone H2AX and Fanconi anemia FANCD2 function in the same pathway to maintain chromosome stability

Fanconi anemia (FA) is a chromosome fragility syndrome characterized by bone marrow failure and cancer susceptibility. The central FA protein FANCD2 is known to relocate to chromatin upon DNA damage in a poorly understood process. Here, we have induced subnuclear accumulation of DNA damage to prove that histone H2AX is a novel component of the FA/BRCA pathway in response to stalled replication forks. Analyses of cells from H2AX knockout mice or expressing a nonphosphorylable H2AX (H2AX(S136A/S139A)) indicate that phosphorylated H2AX (gammaH2AX) is required for recruiting FANCD2 to chromatin at stalled replication forks. FANCD2 binding to gammaH2AX is BRCA1-dependent and cells deficient or depleted of H2AX show an FA-like phenotype, including an excess of chromatid-type chromosomal aberrations and hypersensitivity to MMC. This MMC hypersensitivity of H2AX-deficient cells is not further increased by depleting FANCD2, indicating that H2AX and FANCD2 function in the same pathway in response to DNA damage-induced replication blockage. Consequently, histone H2AX is functionally connected to the FA/BRCA pathway to resolve stalled replication forks and prevent chromosome instability.     

Fanconi anemia proteins stabilize replication forks

Fanconi anemia (FA) is a recessive genetic disorder characterized by hypersensitivity to crosslinking agents that has been attributed to defects in DNA repair and/or replication. FANCD2 and the FA core complex bind to chromatin during DNA replication; however, the role of FA proteins during replication is unknown. Using Xenopus cell-free extracts, we show that FANCL depletion results in defective DNA replication restart following treatment with camptothecin, a drug that results in DSBs during DNA replication. This defect is more pronounced following treatment with mitomycin C, presumably because of an additional role of the FA pathway in DNA crosslink repair. Moreover, we show that chromatin binding of FA core complex proteins during DNA replication follows origin assembly and origin firing and is dependent on the binding of RPA to ssDNA while FANCD2 additionally requires ATR, consistent with FA proteins acting at replication forks. Together, our data suggest that FA proteins play a role in replication restart at collapsed replication forks.     

Human CST complex protects stalled replication forks by directly blocking MRE11 degradation of nascent‐strand DNA

Degradation and collapse of stalled replication forks are main sources of genomic instability, yet the molecular mechanisms for protecting forks from degradation/collapse are not well understood. Here, we report that human CST (CTC1‐STN1‐TEN1) proteins, which form a single‐stranded DNA‐binding complex, localize at stalled forks and protect stalled forks from degradation by the MRE11 nuclease. CST deficiency increases MRE11 binding to stalled forks, leading to nascent‐strand degradation at reversed forks and ssDNA accumulation. In addition, purified CST complex binds to 5’ DNA overhangs and directly blocks MRE11 degradation in vitro, and the DNA‐binding ability of CST is required for blocking MRE11‐mediated nascent‐strand degradation. Our results suggest that CST inhibits MRE11 binding to reversed forks, thus antagonizing excessive nascent‐strand degradation. Finally, we uncover that CST complex inactivation exacerbates genome instability in BRCA2 deficient cells. Collectively, our findings identify the CST complex as an important fork protector that preserves genome integrity under replication perturbation.     

Human Tim/Timeless-interacting protein, Tipin, is required for efficient progression of S phase and DNA replication checkpoint

Tipin was originally isolated as a protein interacting with Timeless/Tim1/Tim (Tim), which is known to be involved in both circadian rhythm and cell cycle checkpoint regulation. The endogenous Tim and Tipin proteins in human cells, interacting through the N-terminal segment of each molecule, form a complex throughout the cell cycle. Tipin and Tim are expressed in the interphase nuclei mostly at constant levels during the cell cycle, and small fractions are recovered in the chromatin-enriched fractions during S phase. Depletion of endogenous Tipin results in reduced growth rate, and this may be due in part to inefficient progression of S phase and DNA synthesis. Knockdown of Tipin induces radioresistant DNA synthesis and inhibits phosphorylation of Chk1 kinase caused by replication stress, as was observed with that of Tim. Knockdown of Tipin or Tim results in reduced protein level and relocation to the cytoplasm of the respective binding partner, suggesting that the complex formation may be required for stabilization and nuclear accumulation of both proteins. Furthermore, both Tipin and Tim may facilitate the accumulation of Claspin in the nuclei under replication stress, whereas nuclear localization of Tipin and Tim is unaffected by Claspin. Our results indicate that mammalian Tipin is a checkpoint mediator that cooperates with Tim and may regulate the nuclear relocation of Claspin in response to replication checkpoint.     

Smc5/6 maintains stalled replication forks in a recombination‐competent conformation

The Smc5/6 structural maintenance of chromosomes complex is required for efficient homologous recombination (HR). Defects in Smc5/6 result in chromosome mis‐segregation and fragmentation. By characterising two Schizosaccharomyces pombe smc6 mutants, we define two separate functions for Smc5/6 in HR. The first represents the previously described defect in processing recombination‐dependent DNA intermediates when replication forks collapse, which leads to increased rDNA recombination. The second novel function defines Smc5/6 as a positive regulator of recombination in the rDNA and correlates mechanistically with a requirement to load RPA and Rad52 onto chromatin genome‐wide when replication forks are stably stalled by nucleotide depletion. Rad52 is required for all HR repair, but Rad52 loading in response to replication fork stalling is unexpected and does not correlate with damage‐induced foci. We propose that Smc5/6 is required to maintain stalled forks in a stable recombination‐competent conformation primed for replication restart.     

Rif1 provides a new DNA‐binding interface for the Bloom syndrome complex to maintain normal replication

BLM, the helicase defective in Bloom syndrome, is part of a multiprotein complex that protects genome stability. Here, we show that Rif1 is a novel component of the BLM complex and works with BLM to promote recovery of stalled replication forks. First, Rif1 physically interacts with the BLM complex through a conserved C‐terminal domain, and the stability of Rif1 depends on the presence of the BLM complex. Second, Rif1 and BLM are recruited with similar kinetics to stalled replication forks, and the Rif1 recruitment is delayed in BLM‐deficient cells. Third, genetic analyses in vertebrate DT40 cells suggest that BLM and Rif1 work in a common pathway to resist replication stress and promote recovery of stalled forks. Importantly, vertebrate Rif1 contains a DNA‐binding domain that resembles the αCTD domain of bacterial RNA polymerase α; and this domain preferentially binds fork and Holliday junction (HJ) DNA in vitro and is required for Rif1 to resist replication stress in vivo. Our data suggest that Rif1 provides a new DNA‐binding interface for the BLM complex to restart stalled replication forks.     

Timeless functions independently of the Tim-Tipin complex to promote sister chromatid cohesion in normal human fibroblasts

The Timeless-Tipin complex and Claspin are mediators of the ATR-dependent activation of Chk1 in the intra-S checkpoint response to stalled DNA replication forks. Tim-Tipin and Claspin also contribute to sister chromatid cohesion (SCC) in various organisms, likely through a replication-coupled process. Some models of the establishment of SCC posit that interactions between cohesin rings and replisomes could result in physiological replication stress requiring fork stabilization. The contributions of Timeless, Tipin, Claspin, Chk1 and ATR to SCC were investigated in genetically stable, human diploid fibroblast cell lines. Whereas Timeless, Tipin and Claspin showed similar contributions to UVC-induced activation of Chk1, siRNA-mediated knockdown of Timeless induced a 100-fold increase in sister chromatid discohesion, whereas the inductive effects of knocking down Tipin, Claspin and ATR were 4–20-fold. Knockdown of Chk1 did not significantly affect SCC. Consistent findings were obtained in two independently derived human diploid fibroblast lines and support a conclusion that SCC in human cells is strongly dependent on Timeless but independent of Chk1. Furthermore, the 10-fold difference in discohesion observed when depleting Timeless versus Tipin indicates that Timeless has a function in SCC that is independent of the Tim-Tipin complex, even though the abundance of Timeless is reduced when Tipin is targeted for depletion. A better understanding of how Timeless, Tipin and Claspin promote SCC will elucidate non-checkpoint functions of these proteins at DNA replication forks and inform models of the establishment of SCC.Key words: cohesion, intra-S checkpoint, Timeless, Tipin, Claspin, ATR, Chk1, human, fibroblast     

Separable functions of Tof1/Timeless in intra-S-checkpoint signalling,replisome stability and DNA topological stress

The highly conserved Tof1/Timeless proteins minimise replication stress and promote normal DNA replication. They are required to mediate the DNA replication checkpoint (DRC), the stable pausing of forks at protein fork blocks, the coupling of DNA helicase and polymerase functions during replication stress (RS) and the preferential resolution of DNA topological stress ahead of the fork. Here we demonstrate that the roles of the Saccharomyces cerevisiae Timeless protein Tof1 in DRC signalling and resolution of DNA topological stress require distinct N and C terminal regions of the protein, whereas the other functions of Tof1 are closely linked to the stable interaction between Tof1 and its constitutive binding partner Csm3/Tipin. By separating the role of Tof1 in DRC from fork stabilisation and coupling, we show that Tof1 has distinct activities in checkpoint activation and replisome stability to ensure the viable completion of DNA replication following replication stress.     

Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity

Swi1 and Swi3 form the replication fork protection complex and play critical roles in proper activation of the replication checkpoint and stabilization of replication forks in the fission yeast Schizosaccharomyces pombe. However, the mechanisms by which the Swi1-Swi3 complex regulates these processes are not well understood. Here, we report functional analyses of the Swi1-Swi3 complex in fission yeast. Swi1 possesses the DDT domain, a putative DNA binding domain found in a variety of chromatin remodeling factors. Consistently, the DDT domain-containing region of Swi1 interacts with DNA in vitro, and mutations in the DDT domain eliminate the association of Swi1 with chromatin in S. pombe cells. DDT domain mutations also render cells highly sensitive to S-phase stressing agents and induce strong accumulation of Rad22-DNA repair foci, indicating that the DDT domain is involved in the activity of the Swi1-Swi3 complex. Interestingly, DDT domain mutations also abolish Swi1's ability to interact with Swi3 in cells. Furthermore, we show that Swi1 is required for efficient chromatin association of Swi3 and that the Swi1 C-terminal domain directly interacts with Swi3. These results indicate that Swi1 associates with chromatin through its DDT domain and recruits Swi3 to function together as the replication fork protection complex.     

The Mi-2/NuRD complex associates with pericentromeric heterochromatin during S phase in rapidly proliferating lymphoid cells

Chromosomal replication results in the duplication not only of DNA sequence but also of the patterns of histone modification, DNA methylation, and nucleoprotein structure that constitute epigenetic information. Pericentromeric heterochromatin in human cells is characterized by unique patterns of histone and DNA modification. Here, we describe association of the Mi-2/NuRD complex with specific segments of pericentromeric heterochromatin consisting of Satellite II/III DNA located on human chromosomes 1, 9, and 16 in some but not all cell types. This association is linked in part to DNA replication and chromatin assembly and may suggest a role in these processes. Mi-2/NuRD accumulation is independent of Polycomb association and is characterized by a unique pattern of histone modification. We propose that Mi-2/NuRD constitutes an enzymatic component of a pathway for assembly and maturation of chromatin utilized by rapidly proliferating lymphoid cells for replication of constitutive heterochromatin. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Timeless functions independently of the Tim-Tipin complex to promote sister chromatid cohesion in normal human fibroblasts

The Timeless-Tipin complex and Claspin are mediators of the ATR-dependent activation of Chk1 in the intra-S checkpoint response to stalled DNA replication forks. Tim-Tipin and Claspin also contribute to sister chromatid cohesion (SCC) in various organisms, likely through a replication-coupled process. Some models of the establishment of SCC posit that interactions between cohesin rings and replisomes could result in physiological replication stress requiring fork stabilization. The contributions of Timeless, Tipin, Claspin, Chk1 and ATR to SCC were investigated in genetically stable, human diploid fibroblast cell lines. Whereas Timeless, Tipin and Claspin showed similar contributions to UVC-induced activation of Chk1, siRNA-mediated knockdown of Timeless induced a 100-fold increase in sister chromatid discohesion, whereas the inductive effects of knocking down Tipin, Claspin and ATR were 4–20-fold. Knockdown of Chk1 did not significantly affect SCC. Consistent findings were obtained in two independently derived human diploid fibroblast lines and support a conclusion that SCC in human cells is strongly dependent on Timeless but independent of Chk1. Furthermore, the 10-fold difference in discohesion observed when depleting Timeless versus Tipin indicates that Timeless has a function in SCC that is independent of the Tim-Tipin complex, even though the abundance of Timeless is reduced when Tipin is targeted for depletion. A better understanding of how Timeless, Tipin and Claspin promote SCC will elucidate non-checkpoint functions of these proteins at DNA replication forks and inform models of the establishment of SCC.     

Mcm10 regulates the stability and chromatin association of DNA polymerase-alpha

Mcm10 is a conserved eukaryotic DNA replication factor whose function has remained elusive. We report here that Mcm10 binding to replication origins in budding yeast is cell cycle regulated and dependent on the putative helicase, Mcm2-7. Mcm10 is also an essential component of the replication fork. A fraction of Mcm10 binds to DNA, as shown by histone association assays that allow for the study of chromatin binding in vivo. However, Mcm10 is also required to maintain steady-state levels of DNA polymerase-alpha (polalpha). In temperature-sensitive mcm10-td mutants, depletion of Mcm10 during S phase results in degradation of the catalytic subunit of polalpha, without affecting other fork components such as Cdc45. We propose that Mcm10 stabilizes polalpha and recruits the complex to replication origins. During elongation, Mcm10 is required for the presence of polalpha at replication forks and may coordinate DNA synthesis with DNA unwinding by the Mcm2-7 complex.     

Exo1 processes stalled replication forks and counteracts fork reversal in checkpoint-defective cells

The replication checkpoint coordinates the cell cycle with DNA replication and recombination, preventing genome instability and cancer. The budding yeast Rad53 checkpoint kinase stabilizes stalled forks and replisome-fork complexes, thus preventing the accumulation of ss-DNA regions and reversed forks at collapsed forks. We searched for factors involved in the processing of stalled forks in HU-treated rad53 cells. Using the neutral-neutral two-dimensional electrophoresis technique (2D gel) and psoralen crosslinking combined with electron microscopy (EM), we found that the Exo1 exonuclease is recruited to stalled forks and, in rad53 mutants, counteracts reversed fork accumulation by generating ss-DNA intermediates. Hence, Exo1-mediated fork processing resembles the action of E. coli RecJ nuclease at damaged forks. Fork stability and replication restart are influenced by both DNA polymerase-fork association and Exo1-mediated processing. We suggest that Exo1 counteracts fork reversal by resecting newly synthesized chains and resolving the sister chromatid junctions that cause regression of collapsed forks.     

Rtt105 functions as a chaperone for replication protein A to preserve genome stability

Generation of single‐stranded DNA (ssDNA) is required for the template strand formation during DNA replication. Replication Protein A (RPA) is an ssDNA‐binding protein essential for protecting ssDNA at replication forks in eukaryotic cells. While significant progress has been made in characterizing the role of the RPA–ssDNA complex, how RPA is loaded at replication forks remains poorly explored. Here, we show that the Saccharomyces cerevisiae protein regulator of Ty1 transposition 105 (Rtt105) binds RPA and helps load it at replication forks. Cells lacking Rtt105 exhibit a dramatic reduction in RPA loading at replication forks, compromised DNA synthesis under replication stress, and increased genome instability. Mechanistically, we show that Rtt105 mediates the RPA–importin interaction and also promotes RPA binding to ssDNA directly in vitro, but is not present in the final RPA–ssDNA complex. Single‐molecule studies reveal that Rtt105 affects the binding mode of RPA to ssDNA. These results support a model in which Rtt105 functions as an RPA chaperone that escorts RPA to the nucleus and facilitates its loading onto ssDNA at replication forks.     

Role for Rif1 in the checkpoint response to damaged DNA in Xenopus egg extracts

TopBP1 is critical for both DNA replication and checkpoint regulation in vertebrate cells. In this study, we have identified Rif1 as a binding partner of TopBP1 in Xenopus egg extracts. In addition, Rif1 also interacts with both ATM and the Mre11-Rad50-Nbs1 (MRN) complex, which are key regulators of checkpoint responses to double-stranded DNA breaks (DSBs). Depletion of Rif1 from egg extracts compromises the activation of Chk1 in response to DSBs but not stalled replication forks. Removal of Rif1 also has a significant impact on the chromatin-binding behavior of key checkpoint proteins. In particular, binding of TopBP1, ATR and the MRN complex to chromatin containing DSBs is reduced in the absence of Rif1. Rif1 interacts with chromatin in a highly regulated and dynamic manner. In unperturbed egg extracts, the association of Rif1 with chromatin depends upon formation of replication forks. In the presence of DSBs, there is elevated accumulation of Rif1 on chromatin under conditions where the activation of ATM is suppressed. Taken together, these results suggest that Rif1 plays a dynamic role in the early steps of a checkpoint response to DSBs in the egg-extract system by promoting the correct accumulation of key regulators on the DNA.