Characterization of factors that underlie transcriptional silencing in C. elegans oocytes.

While it has been appreciated for decades that prophase-arrested oocytes are transcriptionally silenced on a global level, the molecular pathways that promote silencing have remained elusive. Previous work in C. elegans has shown that both topoisomerase II (TOP-2) and condensin II collaborate with the H3K9me heterochromatin pathway to silence gene expression in the germline during L1 starvation, and that the PIE-1 protein silences the genome in the P-lineage of early embryos. Here, we show that all three of these silencing systems, TOP-2/condensin II, H3K9me, and PIE-1, are required for transcriptional repression in oocytes. We find that H3K9me3 marks increase dramatically on chromatin during silencing, and that silencing is under cell cycle control. We also find that PIE-1 localizes to the nucleolus just prior to silencing, and that nucleolar dissolution during silencing is dependent on TOP-2/condensin II. Our data identify both the molecular components and the trigger for genome silencing in oocytes and establish a link between PIE-1 nucleolar residency and its ability to repress transcription.

Delineation of a minimal topoisomerase II binding protein 1 for regulated activation of ATR at DNA double-strand breaks.

Topoisomerase II Binding Protein 1 (TOPBP1) is an important activator of the DNA damage response kinase Ataxia Telangiectasia and Rad3-related (ATR), although the mechanism by which this activation occurs is not yet known. TOPBP1 contains nine copies of the BRCA1 C-terminal repeat (BRCT) motif, which allows protein-protein and protein-DNA interactions. TOPBP1 also contains an ATR activation domain (AAD), which physically interacts with ATR and its partner ATR-interacting protein (ATRIP) in a manner that stimulates ATR kinase activity. It is unclear which of TOPBP1's nine BRCT domains participate in the reaction, as well as the individual roles played by these relevant BRCT domains. To address this knowledge gap, here, we delineated a minimal TOPBP1 that can activate ATR at DNA double-strand breaks in a regulated manner. We named this minimal TOPBP1 "Junior" and we show that Junior is composed of just three regions: BRCT0-2, the AAD, and BRCT7&8. We further defined the individual functions of these three regions by showing that BRCT0-2 is required for recruitment to DNA double-strand breaks and is dispensable thereafter, and that BRCT7&8 is dispensable for recruitment but essential to allow the AAD to multimerize and activate ATR. The delineation of TOPBP1 Junior creates a leaner, simplified, and better understood TOPBP1 and provides insight into the mechanism of ATR activation.

Cyclin CYB-3 controls both S-phase and mitosis and is asymmetrically distributed in the early C. elegans embryo.

In early C. elegans embryos the timing of cell division is both invariant and developmentally regulated, yet how the cell cycle is controlled in the embryo and how cell cycle timing impacts early development remain important, unanswered questions. Here, I focus on the cyclin B3 ortholog CYB-3, and show that this cyclin has the unusual property of controlling both the timely progression through S-phase and mitotic entry, suggesting that CYB-3 is both an S-phase-promoting and mitosis-promoting factor. Furthermore, I find that CYB-3 is asymmetrically distributed in the two-cell embryo, such that the somatic precursor AB cell contains ∼2.5-fold more CYB-3 than its sister cell, the germline progenitor P1 CYB-3 is not only physically limited in P1 but also functionally limited, and this asymmetry is controlled by the par polarity network. These findings highlight the importance of the CYB-3 B3-type cyclin in cell cycle regulation in the early embryo and suggest that CYB-3 asymmetry helps establish the well-documented cell cycle asynchrony that occurs during cell division within the P-lineage.

Eviction notice: new insights into Rad51 removal from DNA during homologous recombination.

In this issue of Molecular Cell, Ward et al. (2010) identify two genes whose products act redundantly to clear Rad51 from DNA after successful strand invasion, thereby enabling the downstream events of homologous recombination to go smoothly.

Direct Binding to Replication Protein A (RPA)-coated Single-stranded DNA Allows Recruitment of the ATR Activator TopBP1 to Sites of DNA Damage.

A critical event for the ability of cells to tolerate DNA damage and replication stress is activation of the ATR kinase. ATR activation is dependent on the BRCT (BRCA1 C terminus) repeat-containing protein TopBP1. Previous work has shown that recruitment of TopBP1 to sites of DNA damage and stalled replication forks is necessary for downstream events in ATR activation; however, the mechanism for this recruitment was not known. Here, we use protein binding assays and functional studies in Xenopus egg extracts to show that TopBP1 makes a direct interaction, via its BRCT2 domain, with RPA-coated single-stranded DNA. We identify a point mutant that abrogates this interaction and show that this mutant fails to accumulate at sites of DNA damage and that the mutant cannot activate ATR. These data thus supply a mechanism for how the critical ATR activator, TopBP1, senses DNA damage and stalled replication forks to initiate assembly of checkpoint signaling complexes.

Regulated proteolysis of DNA polymerase eta during the DNA-damage response in C. elegans.

Both the POLH-1 (pol eta) translesion synthesis (TLS) DNA polymerase and the GEI-17 SUMO E3 ligase are essential for the efficient replication of damaged chromosomes in Caenorhabditis elegans embryos. Here we study how POLH-1 is regulated during a DNA-damage response in these embryos. We report that DNA damage triggers the degradation of POLH-1 and that degradation is mediated by the Cul4-Ddb1-Cdt2 (CRL4-Cdt2) pathway that has previously been shown to degrade the replication factor Cdt1 during S phase. We also show that GEI-17 protects POLH-1 from CRL4-Cdt2-mediated destruction until after it has performed its function in TLS, and this is likely via SUMOylation of POLH-1. These studies reveal that POLH-1 undergoes DNA-damage-induced proteolysis and that GEI-17 regulates the timing of this proteolysis. Implications for how this system may control the removal of POLH-1 from replication forks after TLS are discussed.

Control of DNA replication by the nucleus/cytoplasm ratio in Xenopus.

The nucleus/cytoplasm (N/C) ratio controls S phase dynamics in many biological systems, most notably the abrupt remodeling of the cell cycle that occurs at the midblastula transition in early Xenopus laevis embryos. After an initial series of rapid cleavage cycles consisting only of S and M phases, a critical N/C ratio is reached, which causes a sharp increase in the length of S phase as the cell cycle is reconfigured to resemble somatic cell cycles. How the N/C ratio determines the length of S phase has been a longstanding problem in developmental biology. Using Xenopus egg extracts, we show that DNA replication at high N/C ratio is restricted by one or more limiting substances. We report here that the protein phosphatase PP2A, in conjunction with its B55α regulatory subunit, becomes limiting for replication origin firing at high N/C ratio, and this in turn leads to reduced origin activation and an increase in the time required to complete S phase. Increasing the levels of PP2A catalytic subunit or B55α experimentally restores rapid DNA synthesis at high N/C ratio. Inversely, reduction of PP2A or B55α levels sharply extends S phase even in low N/C extracts. These results identify PP2A-B55α as a link between DNA replication and N/C ratio in egg extracts and suggest a mechanism that may influence the onset of the midblastula transition in vivo.

Transcriptional repression during spermatogenesis in C. elegans requires TOP-2, condensin II, and the MET-2 H3K9 methyltransferase.

In C. elegans RNA polymerase II (RNAPII) transcription is globally repressed as oocytes prepare for meiosis. Recent work has identified the transcriptional repressors responsible for genome silencing in oocytes, and they are topoisomerase II ( TOP-2 ), condensin II, the H3K9 methyltransferase SET-25 and MET-2 , and the PIE-1 protein. Here, we focus on TOP-2 , condensin II, and MET-2 and ask if they play a similar role during spermatogenesis. We report that spermatocytes undergo transcriptional repression, as inferred by a deactivation of RNAPII, and this requires TOP-2 , the CAPG-2 subunit of condensin II, and the histone methyltransferase MET-2 .

NBS1 binds directly to TOPBP1 via disparate interactions between the NBS1 BRCT1 domain and the TOPBP1 BRCT1 and BRCT2 domains.

The TOPBP1 and NBS1 proteins are key components of DNA repair and DNA-based signaling systems. TOPBP1 is a multi-BRCT domain containing protein that plays important roles in checkpoint signaling, DNA replication, and DNA repair. Likewise, NBS1, which is a component of the MRE11-RAD50-NBS1 (MRN) complex, functions in both checkpoint signaling and DNA repair. NBS1 also contains BRCT domains, and previous works have shown that TOPBP1 and NBS1 interact with one another. In this work we examine the interaction between TOPBP1 and NBS1 in detail. We report that NBS1 uses its BRCT1 domain to interact with TOPBP1's BRCT1 domain and, separately, with TOPBP1's BRCT2 domain. Thus, NBS1 can make two distinct contacts with TOPBP1. We report that recombinant TOPBP1 and NBS1 proteins bind one another in a purified system, showing that the interaction is direct and does not require post-translational modifications. Surprisingly, we also report that intact BRCT domains are not required for these interactions, as truncated versions of the domains are sufficient to confer binding. For TOPBP1, we find that small 24-29 amino acid sequences within BRCT1 or BRCT2 allow binding to NBS1, in a transferrable manner. These data expand our knowledge of how the crucial DNA damage response proteins TOPBP1 and NBS1 interact with one another and set the stage for functional analysis of the two disparate binding sites for NBS1 on TOPBP1.

SMK-1/PPH-4.1-mediated silencing of the CHK-1 response to DNA damage in early C. elegans embryos.

During early embryogenesis in Caenorhabditis elegans, the ATL-1-CHK-1 (ataxia telangiectasia mutated and Rad3 related-Chk1) checkpoint controls the timing of cell division in the future germ line, or P lineage, of the animal. Activation of the CHK-1 pathway by its canonical stimulus DNA damage is actively suppressed in early embryos so that P lineage cell divisions may occur on schedule. We recently found that the rad-2 mutation alleviates this checkpoint silent DNA damage response and, by doing so, causes damage-dependent delays in early embryonic cell cycle progression and subsequent lethality. In this study, we report that mutations in the smk-1 gene cause the rad-2 phenotype. SMK-1 is a regulatory subunit of the PPH-4.1 (protein phosphatase 4) protein phosphatase, and we show that SMK-1 recruits PPH-4.1 to replicating chromatin, where it silences the CHK-1 response to DNA damage. These results identify the SMK-1-PPH-4.1 complex as a critical regulator of the CHK-1 pathway in a developmentally relevant context.

MRN-dependent and independent pathways for recruitment of TOPBP1 to DNA double-strand breaks.

Ataxia Telangiectasia mutated and RAD3-related (ATR) kinase is activated by DNA replication stress and also by various forms of DNA damage, including DNA double-strand breaks (DSBs). Recruitment to sites of damage is insufficient for ATR activation as one of two known ATR activators, either topoisomerase II-binding protein (TOPBP1) or Ewing's tumor-associated antigen 1, must also be present for signaling to initiate. Here, we employ our recently established DSB-mediated ATR activation in Xenopus egg extract (DMAX) system to examine how TOPBP1 is recruited to DSBs, so that it may activate ATR. We report that TOPBP1 is only transiently present at DSBs, with a half-life of less than 10 minutes. We also examined the relationship between TOPBP1 and the MRE11-RAD50-NBS1 (MRN), CtBP interacting protein (CtIP), and Ataxia Telangiectasia mutated (ATM) network of proteins. Loss of MRN prevents CtIP recruitment to DSBs, and partially inhibits TOPBP1 recruitment. Loss of CtIP has no impact on either MRN or TOPBP1 recruitment. Loss of ATM kinase activity prevents CtIP recruitment and enhances MRN and TOPBP1 recruitment. These findings demonstrate that there are MRN-dependent and independent pathways that recruit TOPBP1 to DSBs for ATR activation. Lastly, we find that both the 9-1-1 complex and MDC1 are dispensable for TOPBP1 recruitment to DSBs.

Direct requirement for Xmus101 in ATR-mediated phosphorylation of Claspin bound Chk1 during checkpoint signaling.

TopBP1-like proteins, which include Xenopus laevis Xmus101, are required for DNA replication and have been linked to replication checkpoint control. A direct role for TopBP1/Mus101 in checkpoint control has been difficult to prove, however, because of the requirement for replication in generating the DNA structures that activate the checkpoint. Checkpoint activation occurs in X. laevis egg extracts upon addition of an oligonucleotide duplex (AT70). We show that AT70 bypasses the requirement for replication in checkpoint activation. We take advantage of this replication-independent checkpoint system to determine the role of Xmus101 in the checkpoint. We find that Xmus101 is essential for AT70-mediated checkpoint signaling and that it functions to promote phosphorylation of Claspin bound Chk1 by the ataxia-telangiectasia and Rad-3-related (ATR) protein kinase. We also identify a separation-of-function mutant of Xmus101. In extracts expressing this mutant, replication of sperm chromatin occurs normally; however, the checkpoint response to stalled replication forks fails. These data demonstrate that Xmus101 functions directly during signal relay from ATR to Chk1.

Checkpoint silencing during the DNA damage response in Caenorhabditis elegans embryos.

In most cells, the DNA damage checkpoint delays cell division when replication is stalled by DNA damage. In early Caenorhabditis elegans embryos, however, the checkpoint responds to developmental signals that control the timing of cell division, and checkpoint activation by nondevelopmental inputs disrupts cell cycle timing and causes embryonic lethality. Given this sensitivity to inappropriate checkpoint activation, we were interested in how embryos respond to DNA damage. We demonstrate that the checkpoint response to DNA damage is actively silenced in embryos but not in the germ line. Silencing requires rad-2, gei-17, and the polh-1 translesion DNA polymerase, which suppress replication fork stalling and thereby eliminate the checkpoint-activating signal. These results explain how checkpoint activation is restricted to developmental signals during embryogenesis and insulated from DNA damage. They also show that checkpoint activation is not an obligatory response to DNA damage and that pathways exist to bypass the checkpoint when survival depends on uninterrupted progression through the cell cycle.

A global chromatin compaction pathway that represses germline gene expression during starvation.

While much is known about how transcription is controlled at individual genes, comparatively little is known about how cells regulate gene expression on a genome-wide level. Here, we identify a molecular pathway in the C. elegans germline that controls transcription globally in response to nutritional stress. We report that when embryos hatch into L1 larvae, they sense the nutritional status of their environment, and if food is unavailable, they repress gene expression via a global chromatin compaction (GCC) pathway. GCC is triggered by the energy-sensing kinase AMPK and is mediated by a novel mechanism that involves the topoisomerase II/condensin II axis acting upstream of heterochromatin assembly. When the GCC pathway is inactivated, then transcription persists during starvation. These results define a new mode of whole-genome control of transcription.

Structure-function analysis of TOPBP1's role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system.

The protein kinase ATR is activated at sites of DNA double-strand breaks where it plays important roles in promoting DNA end resection and regulating cell cycle progression. TOPBP1 is a multi BRCT repeat containing protein that activates ATR at DSBs. Here we have developed an experimental tool, the DMAX system, to study the biochemical mechanism for TOPBP1-mediated ATR signalling. DMAX combines simple, linear dsDNA molecules with Xenopus egg extracts and results in a physiologically relevant, DSB-induced activation of ATR. We find that DNAs of 5000 nucleotides, at femtomolar concentration, potently activate ATR in this system. By combining immunodepletion and add-back of TOPBP1 point mutants we use DMAX to determine which of TOPBP1's nine BRCT domains are required for recruitment of TOPBP1 to DSBs and which domains are needed for ATR-mediated phosphorylation of CHK1. We find that BRCT1 and BRCT7 are important for recruitment and that BRCT5 functions downstream of recruitment to promote ATR-mediated phosphorylation of CHK1. We also show that BRCT7 plays a second role, independent of recruitment, in promoting ATR signalling. These findings supply a new research tool for, and new insights into, ATR biology.

Ubiquitin/SUMO modification of PCNA promotes replication fork progression in Xenopus laevis egg extracts.

The homotrimeric DNA replication protein proliferating cell nuclear antigen (PCNA) is regulated by both ubiquitylation and sumoylation. We study the appearance and the impact of these modifications on chromosomal replication in frog egg extracts. Xenopus laevis PCNA is modified on lysine 164 by sumoylation, monoubiquitylation, and diubiquitylation. Sumoylation and monoubiquitylation occur during the replication of undamaged DNA, whereas diubiquitylation occurs specifically in response to DNA damage. When lysine 164 modification is prevented, replication fork movement through undamaged DNA slows down and DNA polymerase delta fails to associate with replicating chromatin. When sumoylation alone is prevented, replication occurs normally and neither monoubiquitylation nor sumoylation are required for the replication of simple single-strand DNA templates. Our findings expand the repertoire of functions for PCNA ubiquitylation and sumoylation by elucidating a role for these modifications during the replication of undamaged DNA. Furthermore, they suggest that PCNA monoubiquitylation serves as a molecular gas pedal that controls the speed of replisome movement during S phase.

Biochemical analysis of TOPBP1 oligomerization.

TOPBP1 is an important scaffold protein that helps orchestrate the cellular response to DNA damage. Although it has been previously appreciated that TOPBP1 can form oligomers, how this occurs and the functional consequences for oligomerization were not yet known. Here, we use protein binding assays and other biochemical techniques to study how TOPBP1 self associates. TOPBP1 contains 9 copies of the BRCT domain, and we report that a subset of these BRCT domains interact with one another to drive oligomerization. An intact BRCT 2 domain is required for TOPBP1 oligomerization and we find that the BRCT1&2 region of TOPBP1 interacts with itself and with the BRCT4&5 pair. RAD9 and RHINO are two heterologous binding partners for TOPBP1's BRCT 1&2 domains, and we show that binding of these partners does not come at the expense of TOPBP1 oligomerization. Furthermore, we show that a TOPBP1 oligomer can simultaneously interact with both RAD9 and RHINO. Lastly, we find that the oligomeric state necessary for TOPBP1 to activate the ATR protein kinase is likely to be a tetramer.

DNA damage-induced replication arrest in Xenopus egg extracts.

Chromosomal replication is sensitive to the presence of DNA-damaging alkylating agents, such as methyl methanesulfonate (MMS). MMS is known to inhibit replication though activation of the DNA damage checkpoint and through checkpoint-independent slowing of replication fork progression. Using Xenopus egg extracts, we now report an additional pathway that is stimulated by MMS-induced damage. We show that, upon incubation in egg extracts, MMS-treated DNA activates a diffusible inhibitor that blocks, in trans, chromosomal replication. The downstream effect of the inhibitor is a failure to recruit proliferating cell nuclear antigen, but not DNA polymerase alpha, to the nascent replication fork. Thus, alkylation damage activates an inhibitor that intercepts the replication pathway at a point between the polymerase alpha and proliferating cell nuclear antigen execution steps. We also show that activation of the inhibitor does not require the DNA damage checkpoint; rather, stimulation of the pathway described here results in checkpoint activation. These data describe a novel replication arrest pathway, and they also provide an example of how subpathways within the DNA damage response network are integrated to promote efficient cell cycle arrest in response to damaged DNA.

DNA replication is required for the checkpoint response to damaged DNA in Xenopus egg extracts.

Alkylating agents, such as methyl methanesulfonate (MMS), damage DNA and activate the DNA damage checkpoint. Although many of the checkpoint proteins that transduce damage signals have been identified and characterized, the mechanism that senses the damage and activates the checkpoint is not yet understood. To address this issue for alkylation damage, we have reconstituted the checkpoint response to MMS in Xenopus egg extracts. Using four different indicators for checkpoint activation (delay on entrance into mitosis, slowing of DNA replication, phosphorylation of the Chk1 protein, and physical association of the Rad17 checkpoint protein with damaged DNA), we report that MMS-induced checkpoint activation is dependent upon entrance into S phase. Additionally, we show that the replication of damaged double-stranded DNA, and not replication of damaged single-stranded DNA, is the molecular event that activates the checkpoint. Therefore, these data provide direct evidence that replication forks are an obligate intermediate in the activation of the DNA damage checkpoint.

The Xenopus Xmus101 protein is required for the recruitment of Cdc45 to origins of DNA replication.

The initiation of eukaryotic DNA replication involves origin recruitment and activation of the MCM2-7 complex, the putative replicative helicase. Mini-chromosome maintenance (MCM)2-7 recruitment to origins in G1 requires origin recognition complex (ORC), Cdt1, and Cdc6, and activation at G1/S requires MCM10 and the protein kinases Cdc7 and S-Cdk, which together recruit Cdc45, a putative MCM2-7 cofactor required for origin unwinding. Here, we show that the Xenopus BRCA1 COOH terminus repeat-containing Xmus101 protein is required for loading of Cdc45 onto the origin. Xmus101 chromatin association is dependent on ORC, and independent of S-Cdk and MCM2-7. These results define a new factor that is required for Cdc45 loading. Additionally, these findings indicate that the initiation complex assembly pathway bifurcates early, after ORC association with the origin, and that two parallel pathways, one controlled by MCM2-7, and the other by Xmus101, cooperate to load Cdc45 onto the origin.