A DNA Damage-Regulated BRCT-Containing Protein, TopBP1, Is Required for Cell Survival

BRCA1 carboxyl-terminal (BRCT) motifs are present in a number of proteins involved in DNA repair and/or DNA damage-signaling pathways. Human DNA topoisomerase II binding protein 1 (TopBP1) contains eight BRCT motifs and shares sequence similarity with the fission yeast Rad4/Cut5 protein and the budding yeast DPB11 protein, both of which are required for DNA damage and/or replication checkpoint controls. We report here that TopBP1 is phosphorylated in response to DNA double-strand breaks and replication blocks. TopBP1 forms nuclear foci and localizes to the sites of DNA damage or the arrested replication forks. In response to DNA strand breaks, TopBP1 phosphorylation depends on the ataxia telangiectasia mutated protein (ATM) in vivo. However, ATM-dependent phosphorylation of TopBP1 does not appear to be required for focus formation following DNA damage. Instead, focus formation relies on one of the BRCT motifs, BRCT5, in TopBP1. Antisense Morpholino oligomers against TopBP1 greatly reduced TopBP1 expression in vivo. Similar to that of ataxia telangiectasia-related protein (ATR), Chk1, or Hus1, downregulation of TopBP1 leads to reduced cell survival, probably due to increased apoptosis. Taken together, the data presented here suggest that, like its putative counterparts in yeast species, TopBP1 may be involved in DNA damage and replication checkpoint controls.     

MDC1 collaborates with TopBP1 in DNA replication checkpoint control

Human TopBP1 is a major player in the control of the DNA replication checkpoint. In this study, we identified MDC1, a key checkpoint protein involved in the cellular response to DNA double-strand breaks, as a TopBP1-associated protein. The specific TopBP1-MDC1 interaction is mediated by the fifth BRCT domain of TopBP1 and the Ser-Asp-Thr (SDT) repeats of MDC1. In addition, we demonstrated that TopBP1 accumulation at stalled replication forks is promoted by the H2AX/MDC1 signaling cascade. Moreover, MDC1 is important for ATR-dependent Chk1 activation in response to replication stress. Collectively, our data suggest that MDC1 facilitates several important steps in both cellular DNA damage response and the DNA replication checkpoint.     

Structure of C-terminal tandem BRCT repeats of Rtt107 protein reveals critical role in interaction with phosphorylated histone H2A during DNA damage repair

Rtt107 (regulator of Ty1 transposition 107; Esc4) is a DNA repair protein from Saccharomyces cerevisiae that can restore stalled replication forks following DNA damage. There are six BRCT (BRCA1 C-terminal) domains in Rtt107 that act as binding sites for other recruited proteins during DNA repair. Several Rtt107 binding partners have been identified, including Slx4, Rtt101, Rad55, and the Smc5/6 (structural maintenance of chromosome) protein complex. Rtt107 can reportedly be recruited to chromatin in the presence of Rtt101 and Rtt109 upon DNA damage, but the chromatin-binding site of Rtt107 has not been identified. Here, we report our investigation of the interaction between phosphorylated histone H2A (γH2A) and the C-terminal tandem BRCT repeats (BRCT(5)-BRCT(6)) of Rtt107. The crystal structures of BRCT(5)-BRCT(6) alone and in a complex with γH2A reveal the molecular basis of the Rtt107-γH2A interaction. We used in vitro mutagenesis and a fluorescence polarization assay to confirm the location of the Rtt107 motif that is crucial for this interaction. In addition, these assays indicated that this interaction requires the phosphorylation of H2A. An in vivo phenotypic analysis in yeast demonstrated the critical role of BRCT(5)-BRCT(6) and its interaction with γH2A during the DNA damage response. Our results shed new light on the molecular mechanism by which Rtt107 is recruited to chromatin in response to stalled DNA replication forks.     

Molecular basis of BACH1/FANCJ recognition by TopBP1 in DNA replication checkpoint control

The diverse roles of TopBP1 in DNA replication and checkpoint signaling are associated with the scaffolding ability of TopBP1 to initiate various protein-protein interactions. The recognition of the BACH1/FANCJ helicase by TopBP1 is critical for the activation of the DNA replication checkpoint at stalled replication forks and is facilitated by the C-terminal tandem BRCT7/8 domains of TopBP1 and a phosphorylated Thr(1133) binding motif in BACH1. Here we provide the structural basis for this interaction through analysis of the x-ray crystal structures of TopBP1 BRCT7/8 both free and in complex with a BACH1 phospho-peptide. In contrast to canonical BRCT-phospho-peptide recognition, TopBP1 BRCT7/8 undergoes a dramatic conformational change upon BACH1 binding such that the two BRCT repeats pivot about the central BRCT-BRCT interface to provide an extensive and deep peptide-binding cleft. Additionally, we provide the first structural mechanism for Thr(P) recognition among BRCT domains. Together with systematic mutagenesis studies, we highlight the role of key contacts in governing the unique specificity of the TopBP1-BACH1 interaction.     

TopBP1 localises to centrosomes in mitosis and to chromosome cores in meiosis

Topoisomerase IIbeta binding protein 1 (TopBP1), previously shown to localise to sites of DNA damage and to stalled replication forks, has been implicated in DNA replication and in DNA damage response. In this work we showed that TopBP1 was localised in structures other than stalled replication forks. In late mitosis TopBP1 localises to centrosomes in a manner similar to other DNA damage response proteins such as BRCA1 and p53. Spindle checkpoint activation does not affect this centrosomal localisation. Moreover, in the testis, we detected high levels of TopBP1 associated with meiotic prophase chromosome cores and the X-Y pair. Together, these data suggest a direct role of TopBP1 during both mitosis and meiotic prophase I.     

BRCT domain-containing protein TopBP1 functions in DNA replication and damage response

Topoisomerase IIbeta-binding protein (TopBP1), a human protein with eight BRCT domains, is similar to Saccharomyces cerevisiae Dpb11 and Schizosaccharomyces pombe Cut5 checkpoint proteins and closely related to Drosophila Mus101. We show that human TopBP1 is required for DNA replication and that it interacts with DNA polymerase epsilon. In S phase TopBP1 colocalizes with Brca1 to foci that do not represent sites of ongoing DNA replication. Inhibition of DNA synthesis leads to relocalization of TopBP1 together with Brca1 to replication forks, suggesting a role in rescue of stalled forks. DNA damage induces formation of distinct TopBP1 foci that colocalize with Brca1 in S phase, but not in G(1) phase. We also show that TopBP1 interacts with the checkpoint protein hRad9. Thus, these results implicate TopBP1 in replication and checkpoint functions.     

The DNA repair component Metnase regulates Chk1 stability

Chk1 both arrests replication forks and enhances repair of DNA damage by phosphorylation of downstream effectors. Metnase (also termed SETMAR) is a SET histone methylase and transposase nuclease protein that promotes both DNA double strand break (DSB) repair and re-start of stalled replication forks. We previously found that Chk1 phosphorylation of Metnase on S495 enhanced its DNA DSB repair activity but decreased its ability to re-start stalled replication forks. Here we show that phosphorylated Metnase feeds back to increase the half-life of Chk1. Chk1 half-life is regulated by DDB1 targeting it to Cul4A for ubiquitination and destruction. Metnase decreases Chk1 interaction with DDB1, and decreases Chk1 ubiquitination. These data define a novel pathway for Chk1 regulation, whereby a target of Chk1, Metnase, feeds back to amplify Chk1 stability, and therefore enhance replication fork arrest.     

MCPH1/BRIT1 cooperates with E2F1 in the activation of checkpoint, DNA repair and apoptosis

Microcephalin (MCPH1) has a crucial role in the DNA damage response by promoting the expression of Checkpoint kinase 1 (CHK1) and Breast cancer susceptibility gene 1 (BRCA1); however, the mechanism of this regulation remains unclear. Here, we show that MCPH1 regulates CHK1 and BRCA1 through the interaction with E2F1 on the promoters of both genes. MCPH1 also regulates other E2F target genes involved in DNA repair and apoptosis such as RAD51, DDB2, TOPBP1, p73 and caspases. MCPH1 interacts with E2F1 on the p73 promoter, and regulates p73 induction and E2F1-induced apoptosis as a result of DNA damage. MCPH1 forms oligomers through the second and third BRCT domains. An MCPH1 mutant containing only its oligomerization domain has a dominant-negative role by blocking MCPH1 binding to E2F1. It also inhibits p73 induction in DNA damage and E2F1-dependent apoptosis. Taken together, MCPH1 cooperates with E2F1 to regulate genes involved in DNA repair, checkpoint and apoptosis, and might participate in the maintenance of genomic integrity.     

The DNA topoisomerase IIbeta binding protein 1 (TopBP1) interacts with poly (ADP-ribose) polymerase (PARP-1)

We investigated the physical association of the DNA topoisomerase IIbeta binding protein 1 (TopBP1), involved in DNA replication and repair but also in regulation of apoptosis, with poly(ADP-ribose) polymerase-1 (PARP-1). This enzyme plays a crucial role in DNA repair and interacts with many DNA replication/repair factors. It was shown that the sixth BRCA1 C-terminal (BRCT) domain of TopBP1 interacts with a protein fragment of PARP-1 in vitro containing the DNA-binding and the automodification domains. More significantly, the in vivo interaction of endogenous TopBP1 and PARP-1 proteins could be shown in HeLa-S3 cells by co-immunoprecipitation. TopBP1 and PARP-1 are localized within overlapping regions in the nucleus of HeLa-S3 cells as shown by immunofluorescence. Exposure to UVB light slightly enhanced the interaction between both proteins. Furthermore, TopBP1 was detected in nuclear regions where poly(ADP-ribose) (PAR) synthesis takes place and is ADP-ribosylated by PARP-1. Finally, cellular (ADP-ribosyl)ating activity impairs binding of TopBP1 to Myc-interacting zinc finger protein-1 (Miz-1). The results indicate an influence of post-translational modifications of TopBP1 on its function during DNA repair.     

Cockayne syndrome group B protein regulates fork restart,fork progression and MRE11-dependent fork degradation in BRCA1/2-deficient cells

Cockayne syndrome group B (CSB) protein has been implicated in the repair of a variety of DNA lesions that induce replication stress. However, little is known about its role at stalled replication forks. Here, we report that CSB is recruited to stalled forks in a manner dependent upon its T1031 phosphorylation by CDK. While dispensable for MRE11 association with stalled forks in wild-type cells, CSB is required for further accumulation of MRE11 at stalled forks in BRCA1/2-deficient cells. CSB promotes MRE11-mediated fork degradation in BRCA1/2-deficient cells. CSB possesses an intrinsic ATP-dependent fork reversal activity in vitro, which is activated upon removal of its N-terminal region that is known to autoinhibit CSB’s ATPase domain. CSB functions similarly to fork reversal factors SMARCAL1, ZRANB3 and HLTF to regulate slowdown in fork progression upon exposure to replication stress, indicative of a role of CSB in fork reversal in vivo. Furthermore, CSB not only acts epistatically with MRE11 to facilitate fork restart but also promotes RAD52-mediated break-induced replication repair of double-strand breaks arising from cleavage of stalled forks by MUS81 in BRCA1/2-deficient cells. Loss of CSB exacerbates chemosensitivity in BRCA1/2-deficient cells, underscoring an important role of CSB in the treatment of cancer lacking functional BRCA1/2.     

Mcm10 interacts with Rad4/Cut5(TopBP1) and its association with origins of DNA replication is dependent on Rad4/Cut5(TopBP1)

Initiation of DNA replication in eukaryotes is a highly conserved and ordered process involving the co-ordinated, stepwise association of distinct proteins at multiple origins of replication throughout the genome. Here, taking Schizosaccharomyces pombe as a model, the role of Rad4(TopBP1) in the assembly of the replication complex has been examined. Quantitative chromatin immunoprecipitation experiments confirm that Rad4(TopBP1) associates with origins of DNA replication and, in addition, demonstrate that the protein is not present within the active replisome. A direct interaction between Rad4(TopBP1) and Mcm10 is shown and this is reflected in the Rad4(TopBP1)-dependent origin association of Mcm10. Rad4(TopBP1) is also shown to interact with Sld2 and Sld3 and to be required for the stable origin association of these two proteins. Rad4(TopBP1) chromatin association at stalled replication forks was found to be dependent upon the checkpoint protein Rad9, which was not required for Rad4(TopBP1) origin association. Comparison of the levels of chromatin association at origins of replication and stalled replication forks and the differential requirement for Rad9 suggest functional differences for Rad4(TopBP1) at these distinct sites.     

How Cells Activate ATR

ATR is a critical upstream regulator of checkpoint responses to incompletely replicated and damaged DNA. However, it had not been understood how the kinase activity of ATR is switched on during checkpoint responses. TopBP1 and its homologs are necessary for both DNA replication and checkpoint control. A recent report from this laboratory demonstrated that TopBP1 functions as an activator of ATR. It had been known that TopBP1 accumulates at sites of replicative stress and DNA damage. Thus, interaction of ATR with a critical protein at stalled replication forks and sites of DNA damage triggers its activation. This finding helps to explain how aberrant DNA structures in the genome induce ATR-dependent signaling processes.     

The Rev1 translesion synthesis polymerase has multiple distinct DNA binding modes

Rev1 is a eukaryotic DNA polymerase of the Y family involved in translesion synthesis (TLS), a major damage tolerance pathway that allows DNA replication at damaged templates. Uniquely amongst the Y family polymerases, the N-terminal part of Rev1, dubbed the BRCA1 C-terminal homology (BRCT) region, includes a BRCT domain. While most BRCT domains mediate protein-protein interactions, Rev1 contains a predicted α-helix N-terminal to the BRCT domain and in human Replication Factor C (RFC) such a BRCT region endows the protein with DNA binding capacity. Here, we studied the DNA binding properties of yeast and mouse Rev1. Our results show that the BRCT region of Rev1 specifically binds to a 5' phosphorylated, recessed, primer-template junction. This DNA binding depends on the extra α-helix, N-terminal to the BRCT domain. Surprisingly, a stretch of 20 amino acids N-terminal to the predicted α-helix is also critical for high-affinity DNA binding. In addition to 5' primer-template junction binding, Rev1 efficiently binds to a recessed 3' primer-template junction. These dual DNA binding characteristics are discussed in view of the proposed recruitment of Rev1 by 5' primer-template junctions, downstream of stalled replication forks.     

The Mre11-Rad50-Nbs1 complex acts both upstream and downstream of ataxia telangiectasia mutated and Rad3-related protein (ATR) to regulate the S-phase checkpoint following UV treatment

The Mre11-Rad50-Nbs1 (MRN) complex is required for mediating the S-phase checkpoint following UV treatment, but the underlying mechanism is not clear. Here we demonstrate that at least two mechanisms are involved in regulating the S-phase checkpoint in an MRN-dependent manner following UV treatment. First, when replication forks are stalled, MRN is required upstream of ataxia telangiectasia mutated and Rad3-related protein (ATR) to facilitate ATR activation in a substrate and dosage-dependent manner. In particular, MRN is required for ATR-directed phosphorylation of RPA2, a critical event in mediating the S-phase checkpoint following UV treatment. Second, MRN is a downstream substrate of ATR. Nbs1 is phosphorylated by ATR at Ser-343 when replication forks are stalled, and this phosphorylation event is also important for down-regulating DNA replication following UV treatment. Moreover, we demonstrate that MRN and ATR/ATR-interacting protein (TRIP) interact with each other, and the forkhead-associated/breast cancer C-terminal domains (FHA/BRCT) of Nbs1 play a significant role in mediating this interaction. Mutations in the FHA/BRCT domains do not prevent ATR activation but specifically impair ATR-mediated Nbs1 phosphorylation at Ser-343, which results in a defect in the S-phase checkpoint. These data suggest that MRN plays critical roles both upstream and downstream of ATR to regulate the S-phase checkpoint when replication forks are stalled.     

Regulation of DNA replication through Sld3-Dpb11 interaction is conserved from yeast to humans

Cyclin-dependent kinases (CDKs) play crucial roles in promoting DNA replication and preventing rereplication in eukaryotic cells [1-4]. In budding yeast, CDKs promote DNA replication by phosphorylating two proteins, Sld2 and Sld3, which generates binding sites for pairs of BRCT repeats (breast cancer gene 1 [BRCA1] C terminal repeats) in the Dpb11 protein [5, 6]. The Sld3-Dpb11-Sld2 complex generated by CDK phosphorylation is required for the assembly and activation of the Cdc45-Mcm2-7-GINS (CMG) replicative helicase. In response to DNA replication stress, the interaction between Sld3 and Dpb11 is blocked by the checkpoint kinase Rad53 [7], which prevents late origin firing [7, 8]. Here we show that the two key CDK sites in Sld3 are conserved in the human Sld3-related protein Treslin/ticrr and are essential for DNA replication. Moreover, phosphorylation of these two sites mediates interaction with the orthologous pair of BRCT repeats in the human Dpb11 ortholog, TopBP1. Finally, we show that DNA replication stress prevents the interaction between Treslin/ticrr and TopBP1 via the Chk1 checkpoint kinase. Our results indicate that Treslin/ticrr is a genuine ortholog of Sld3 and that the Sld3-Dpb11 interaction has remained a critical nexus of S phase regulation through eukaryotic evolution.     

Chk1 inhibition after replicative stress activates a double strand break response mediated by ATM and DNA-dependent protein kinase

Checkpoint kinase 1 (Chk1) regulates cell cycle checkpoints and DNA damage repair in response to genotoxic stress. Inhibition of Chk1 is an emerging strategy for potentiating the cytotoxicity of chemotherapeutic drugs. Here, we demonstrate that AZD7762, an ATP-competitive Chk1/2 inhibitor induces γ-H2AX in gemcitabine-treated cells by altering both dynamics and stability of replication forks, allowing the firing of suppressed replication origins as measured by DNA fiber combing and causing a dramatic increase in DNA breaks as measured by comet assay. Furthermore, we identify ATM and DNA-PK, rather than ATR, as the kinases mediating γ-H2AX induction, suggesting AZD7762 converts stalled forks into double strand breaks (DSBs). Consistent with DSB formation upon fork collapse, cells deficient in DSB repair by lacking BRCA2, XRCC3, or DNA-PK were selectively more sensitive to combined AZD7762 and gemcitabine. Checkpoint abrogation by AZD7762 also caused premature mitosis in gemcitabine-treated cells arrested in G1/early S-phase. Prevention of premature mitotic entry via Cdk1 siRNA knockdown suppressed apoptosis. These results demonstrate that chemosensitization of gemcitabine by Chk1 inhibition results from at least three cellular events namely activation of origin firing, destabilization of stalled replication forks, and entry of cells with damaged DNA into lethal mitosis. Additionally, the current study indicates that the combination of Chk1 inhibitor and gemcitabine may be particularly effective in targeting tumors with specific DNA repair defects.