Topoisomerase II Binding Protein 1 Research Articles

Evolution of the triplet BRCT domain

Organisms have evolved a complex system, called the DNA damage response (DDR), which maintains genome integrity. The DDR is responsible for identifying and repairing a variety of lesions and alterations in DNA. DDR proteins coordinate DNA damage detection, cell cycle arrest, and repair, with many of these events regulated by protein phosphorylation. In the human proteome, 23 proteins contain the BRCT (BRCA1 C‐Terminus domain) domain, a modular signaling domain that can bind phosphopeptides and mediate protein-protein interactions. BRCTs can be found as functional single units, tandem (tBRCT), triplet (tpBRCT), and quartet. Here we examine the evolution of the tpBRCT architecture present in TOPBP1 (DNA topoisomerase II binding protein 1) and ECT2 (epithelial cell transforming 2), and their respective interaction partners RAD9 (Cell cycle checkpoint control protein RAD9) and CYK-4 (Rac GTPase-activating protein 1), with a focus on the conservation of the phosphopeptide-binding residues. The pair TOPBP1-RAD9 arose with the Eukaryotes and ECT2-CYK-4 with the Eumetazoans. Triplet structural and functional characteristics were conserved in almost all organisms. The first unit of the triplet (BRCT0) is different from the other two BRCTs but conserved between orthologs for both TOPBP1 and ECT2. BRCT domain evolution simulations suggest a trend to retain the singlet or towards two or three BRCT copies per protein consistent with functional tBRCT and tpBRCT architectures. Our results shed light on the emergence of the function and architecture of multiple BRCT domain organizations and provide information about the evolution of the BRCT triplet. Knowledge of BRCT domain evolution can improve the understanding of DNA damage response mechanisms and signal transduction in DDR.

Mitotic tethering enables inheritance of shattered micronuclear chromosomes.

Chromothripsis, the shattering and imperfect reassembly of one (or a few) chromosome(s)1, is an ubiquitous2 mutational process generating localized and complex chromosomal rearrangements that drive genome evolution in cancer. Chromothripsis can be initiated by mis-segregation errors in mitosis3,4 or DNA metabolism5-7 that lead to entrapment of chromosomes within micronuclei and their subsequent fragmentation in the next interphase or following mitotic entry6,8-10. Here we use inducible degrons to demonstrate that chromothriptically produced pieces of a micronucleated chromosome are tethered together in mitosis by a protein complex consisting of mediator of DNA damage checkpoint 1(MDC1), DNA topoisomerase II-binding protein 1(TOPBP1) and cellular inhibitor of PP2A (CIP2A), thereby enabling en masse segregation to the same daughter cell. Such tethering is shown to be crucial for the viability of cells undergoing chromosome mis-segregation and shattering after transient inactivation of the spindle assembly checkpoint. Transient, degron-induced reduction in CIP2A following chromosome micronucleation-dependent chromosome shattering is shown to drive acquisition of segmental deletions and inversions. Analyses of pancancer tumour genomes showed that expression of CIP2A and TOPBP1 was increased overallin cancers withgenomic rearrangements, including copy number-neutralchromothripsiswith minimal deletions, but comparatively reduced in cancers with canonical chromothripsis in which deletions were frequent. Thus, chromatin-bound tethers maintain the proximity of fragments of a shattered chromosome enabling their re-encapsulation into, and religation within, a daughter cell nucleus to form heritable, chromothriptically rearranged chromosomes found in the majority of human cancers.

Convergence of SIRT1 and ATR signaling to modulate replication origin dormancy.

During routine genome duplication, many potential replication origins remain inactive or 'dormant'. Such origin dormancy is achieved, in part, by an interaction with the metabolic sensor SIRT1 deacetylase. We report here that dormant origins are a group of consistent, pre-determined genomic sequences that are distinguished from baseline (i.e. ordinarily active) origins by their preferential association withtwo phospho-isoforms of the helicase component MCM2.During normal unperturbed cell growth, baseline origins, but not dormant origins, associate with a form of MCM2 that is phosphorylated by DBF4-dependent kinase (DDK) on serine 139 (pS139-MCM2). This association facilitates the initiation of DNA replication from baseline origins. Concomitantly, SIRT1 inhibits Ataxia Telangiectasia and Rad3-related (ATR)-kinase-mediated phosphorylation of MCM2 on serine 108 (pS108-MCM2) by deacetylating the ATR-interacting protein DNA topoisomerase II binding protein 1 (TOPBP1), thereby preventing ATR recruitment to chromatin.In cells devoid of SIRT1 activity, or challenged by replication stress, this inhibition is circumvented, enabling ATR-mediated S108-MCM2 phosphorylation.In turn, pS108-MCM2 enables DDK-mediated phosphorylation on S139-MCM2 and facilitates replication initiation at dormant origins. These observations suggest that replication origin dormancy and activation are regulated by distinct post-translational MCM modifications that reflect a balance between SIRT1 activity and ATR signaling.

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.

EP159: A TOPBP1 variant associated With BAFopathy methylation pattern

DNA Topoisomerase II Binding Protein 1 (TOPBP1) is a topoisomerase involved in maintaining genomic integrity via numerous functions including the regulation of DNA replication and replication stress response. A number of studies have shown that TOPBP1 also interacts with the BAF complex. The BAF complex is a group of ATP-dependent chromatin remodeling enzymes, including 10-12 proteins that regulate the nucleosomal structure by altering histone-DNA interactions. For example, the BRCT motif of TOPBP1 interacts with E2F1, a transcription factor, by recruiting BRG1/BRM, a core member of the BAF complex, to prevent apoptosis. In addition, TOPBP1 directly associates with BRG1(SMARCA4) and BAF250a (ARID1A) in the BAF complex to prevent DNA entanglement during mitosis. While TOPBP1 is not currently associated with a genetic syndrome, the BAF complex proteins with which it interacts are associated with BAFopathies such as Coffin Siris Syndrome (CSS) and Nicolaides-Baraitser Syndrome (NCBRS). For example, the gene encoding BRG1 (SMARCA4) is mutated in 11% of individuals affected by CSS, and the gene encoding BRM (SMARCA2) is mutated in almost all NCBRS patients. The gene encoding BAF250a (ARID1A) is also associated with CSS. We have previously demonstrated overlapping DNA methylation episignatures in individuals with CSS and NCBRS, consistent with a common functional basis of these disorders. A 15-month-old boy, born to first cousin Syrian parents, presented with respiratory distress, recurrent aspiration pneumonia, failure to thrive, seizure disorder, cortical blindness, microcephaly, and severe developmental delay. He was born at term weighing 2.2 kg and started having seizures at 4 months. At 8 months, after his first aspiration pneumonia, he stopped sucking and swallowing. At 15 months, he was very small with weight, length, and head circumference at a 4 to 8 month level. He made few attempts to move spontaneously and had no head control. He did not fix or follow, showed no blinking response to threat, and had disconjugate and roving eye movements. There was no response to sounds. He had a right plagiocephaly, deficiency of the lateral eyebrows, high arched palate, and long ears. No organomegaly was noted. He had significant central hypotonia with head lag. There was spasticity with flexion contractures in the upper extremities. Lower extremities had normal tone with hyperreflexia. He had intermittent spontaneous hip flexion. Family history was positive for a sister who developed seizures at 8 months, had poor vision and developmental delay, and died at 7 years. A brother has mild to moderate intellectual delay with no history of seizures or poor vision. MRI of the head showed severe cerebral atrophy with nonobstructive hydrocephalus ex-vacuo. An echocardiogram showed moderate aortic root dilatation. Metabolic testing was negative. Chromosome microarray was normal. Exome sequencing with trio analysis showed a de novo variant of uncertain significance in a candidate gene, NM_007027.3(TOPBP1):c.2396C>T, p.(Ala799Val). He was also hemizygous for a maternally inherited VUS in MAP7D2 and heterozygous for a paternally inherited VUS in TGFBR3. DNA from peripheral blood was analyzed by EpiSign version 3 which includes an episignature for BAFopathies based on pathogenic variants in ARID1A, ARID1B, SMARCB1, SMARCA4, and SMARCA2. Results showed a methylation pattern which was inconclusive with mild methylation differences overlapping the episignature for CSS and NCBRS. There is an overlap between this patient’s phenotype and the features of CSS and NCBRS. For example, CSS and NCBRS can present with variable degrees of developmental delay, seizures, feeding difficulties, hypotonia, visual deficits, slow growth, and microcephaly. NCBRS can also present with regression or lack of developmental progress, especially at the onset of seizures, and CSS is associated with frequent upper respiratory tract infections. In this patient with a VUS in TOPBP1, the overlap of methylation profile with CSS and NCBRS is intriguing, given the known interaction of TOPBP1 with the BAF complex. We propose that the child’s clinical features and methylation findings may be due to a disruption of the TOPBP1-BAF interaction and that the TOPBP1 variant is actually pathogenic. Since there is currently no known association between TOPBP1 and any genetic syndrome, we suggest that any cases with TOPBP1 variants identified through exome sequencing be assessed for the episignature associated with the BAFopathies.

Non-canonical role of the ATR pathway in axon regeneration as a mechanosensitive brake.

Non-canonical role of the ATR pathway in axon regeneration as a mechanosensitive brake.

Suppression of TopBP1 function increases the efficacy of chemotherapeutic treatments by enhancing the induction of apoptosis

AbstractThe DNA damage response (DDR) is an important mechanism to maintain genome integrity by arresting cell cycle progression and inducing DNA repair and/or apoptosis. Therefore, the activity of DDR is closely related to the drug sensitivity of cancer cells. Inhibitors of ATR, a key member of protein kinases functioning in DDR, are attractive candidates as sensitizers in chemotherapy. In this study, we explore another candidate of chemosensitizers and report DNA topoisomerase II binding protein 1 (TopBP1), a regulator of ATR‐mediated signaling, as a potential target to increase the efficacy of chemotherapeutic treatments. Suppression of TopBP1 using siRNA increased cancer cell sensitivity to cisplatin and an alkylating agent N‐methyl‐N‐nitrosourea (MNU), concomitant with a percentage increase in the sub‐G1 population and caspase‐9 activation. The immunoblotting analysis revealed that the phosphorylation of CHK1 was significantly reduced in TopBP1‐knockdown cells. Consequently, treatment with an ATR inhibitor dramatically increased the production of the sub‐G1 population compared to an ATM inhibitor. Phosphorylation of RPA2 increased after drug treatment in TopBP1‐knockdown cells. These results suggest that TopBP1 is involved in DDR protecting stalled forks from collapse and preventing apoptosis through the activation of an ATR/CHK1 signaling pathway.

HTATSF1-Nucleated PARylation and Phosphorylation Cascade Dictates Homologous Recombination and Modulates Chemotherapeutic Response of Breast Carcinoma

Homologous recombination (HR) is a high-fidelity, template-dependent repair pathway of DNA double-strand breaks (DSBs). The efficiency of HR system is tightly associated with tumor response to chemotherapy. Yet, how HR activity is regulated remains to be investigated. Here, we report that the RNA binding protein HTATSF1 is physically associated with Topoisomerase II-binding protein 1 (TOPBP1) in a cell-cycle and phosphorylation dependent manner. In specific, S-phase enriched HTATSF1 is phosphorylated by CK2 kinase at Ser748 and the phosphorylation mark is read by the N-terminal BRCT domains of TOPBP1. Importantly, the RNA recognition motifs (RRMs) of HTATSF1 interact with poly(ADP-ribosyl)ated RPA, and this allows the engagement of HTATSF1-TOPBP1 complex to post-resected DSB ends and facilitates the exchange of RPA and RAD51 to favor HR repair of DSBs. Furthermore, we showed CK2-HTATSF1-TOPBP1 axis is generally hyperactivated in multiple malignancies and imparts chemo-insensitivity of breast tumor, while loss-of-function mutation of HTATSF1 in clinical tumors predicts higher HR deficiency (HRD) scores and elicits vulnerabilities of tumor cells to PARP inhibitors (PARPis) or platinum drug. Together, our study reveals an S-phase specific HTATSF1-nucleated PARylation and phosphorylation cascade that directs the HR-promoting activity of TOPBP1, and suggests that the integrity of CK2-HTATSF1-TOPBP1 axis may serve as an indicator of tumor HR status and modulate chemotherapeutic response .

The synergistic proapoptotic effect of PARP-1 and HDAC inhibition in cutaneous T-cell lymphoma is mediated via Blimp-1

AbstractThe therapy of advanced mycosis fungoides (MF) presents a therapeutic challenge, and the search for new therapeutic targets is ongoing. Poly(ADP-ribose) polymerase 1 was shown to be upregulated in patients with advanced MF and could be druggable by a new class of chemotherapeutic agents, PARP-1 inhibitors, which are already in clinical trials for other malignancies; however, the role of PARP-1 inhibitors in MF has never been established. We examined the efficacy of talazoparib in the murine model of cutaneous T-cell lymphoma. The cytotoxic effect of talazoparib on Moloney MuLV-induced T-cell lymphoma (MBL2) cells was a result of G2/M cell cycle arrest via the upregulation of p53. The in vivo experiments confirmed the clinical impact of talazoparib on MF tumors. When talazoparib was combined with the histone deacetylase (HDAC) inhibitor, romidepsin, the cytotoxic effect was synergized via downregulation of the DNA-repair genes Fanconianemia complementation group A (FANCA), Fanconi anemia complementation group D2 (FANCD2), and DNA topoisomerase II binding protein 1(TOPBP1)and stimulation of apoptosis via Blimp-1 (PRDM1)/Bax axis. Romidepsin increased the expression of IRF8and Bcl-6, leading to upregulation of Blimp1and Bax; whereas talazoparib upregulated Blimp-1 and Bax via upregulation of interferon regulatory factor 4 (IRF4), leading to cleavage of caspases 6 and 7. Thus, a combination of talazoparib with romidepsin demonstrated the synergistic antilymphoma effect and warranted further investigation in a clinical trial.

Topoisomerase II-binding protein 1 promotes the progression of prostate cancer via ATR-CHK1 signaling pathway.

DNA damage response (DDR) plays an important role in the progression of cancers, including prostate cancer (PCa). Topoisomerase II-binding protein 1 (TopBP1) is an essential promotor of ATR-mediated DDR. Herein, we investigated the association between TopBP1 and PCa and determined its effect on the progression of PCa. The expression and clinical features of TopBP1 were analyzed using large-scale cohort of tissue microarray analyses and The Cancer Genome Atlas database, which indicated that TopBP1 was positively correlated with high Gleason Score, advanced clinical and pathological stages, the metastasis status. Multivariate analysis revealed that the upregulation of TopBP1 was an independent predictor for a worse biochemical recurrence-free survival (BCR-free survival). Furthermore, we discovered that downregulation of TopBP1 significantly suppressed the growth and migration ability of PCa lines by loss-of-function assays in vitro. Further mechanistic investigations clarified that TopBP1 promoted proliferation and migration by activating ATR-Chk1 signaling pathway.

Common motifs in ETAA1 and TOPBP1 required for ATR kinase activation

DNA damage response Ser/Thr kinases, including ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR), control cell cycle progression, DNA repair, and apoptosis. ATR is activated by ETAA1 activator of ATR kinase (ETAA1) or DNA topoisomerase II binding protein 1 (TOPBP1). Both ETAA1 and TOPBP1 contain experimentally defined ATR activation domains (AADs) that are mostly unstructured and have minimal sequence similarity. A tryptophan residue in both AADs is required for ATR activation, but the other features of these domains and the mechanism by which they activate ATR are unknown. In this study, using bioinformatic analyses, kinase assays, co-immunoprecipitation, and immunofluorescence measures of signaling, we more specifically defined the TOPBP1 and ETAA1 AADs and identified additional features of the AADs needed for ATR activation. We found that both ETAA1 and TOPBP1 contain a predicted coiled-coil motif that is required for ATR activation in vitro and in cells. Mutation of the predicted coiled coils does not alter AAD oligomerization but does impair binding of the AADs to ATR. These results suggest that TOPBP1 and ETAA1 activate ATR using similar motifs and mechanisms.

The Cdk8/19-cyclin C transcription regulator functions in genome replication through metazoan Sld7

Accurate genome duplication underlies genetic homeostasis. Metazoan Mdm2 binding protein (MTBP) forms a main regulatory platform for origin firing together with Treslin/TICRR and TopBP1 (Topoisomerase II binding protein 1 (TopBP1)–interacting replication stimulating protein/TopBP1-interacting checkpoint and replication regulator). We report the first comprehensive analysis of MTBP and reveal conserved and metazoa-specific MTBP functions in replication. This suggests that metazoa have evolved specific molecular mechanisms to adapt replication principles conserved with yeast to the specific requirements of the more complex metazoan cells. We uncover one such metazoa-specific process: a new replication factor, cyclin-dependent kinase 8/19–cyclinC (Cdk8/19-cyclin C), binds to a central domain of MTBP. This interaction is required for complete genome duplication in human cells. In the absence of MTBP binding to Cdk8/19-cyclin C, cells enter mitosis with incompletely duplicated chromosomes, and subsequent chromosome segregation occurs inaccurately. Using remote homology searches, we identified MTBP as the metazoan orthologue of yeast synthetic lethal with Dpb11 7 (Sld7). This homology finally demonstrates that the set of yeast core factors sufficient for replication initiation in vitro is conserved in metazoa. MTBP and Sld7 contain two homologous domains that are present in no other protein, one each in the N and C termini. In MTBP the conserved termini flank the metazoa-specific Cdk8/19-cyclin C binding region and are required for normal origin firing in human cells. The N termini of MTBP and Sld7 share an essential origin firing function, the interaction with Treslin/TICRR or its yeast orthologue Sld3, respectively. The C termini may function as homodimerisation domains. Our characterisation of broadly conserved and metazoa-specific initiation processes sets the basis for further mechanistic dissection of replication initiation in vertebrates. It is a first step in understanding the distinctions of origin firing in higher eukaryotes.

Topoisomerase II binding protein 1 (TOPBP1)

Topoisomerase II binding protein 1 (TOPBP1)

Rad9A Is Required for G2 Decatenation Checkpoint and to Prevent Endoreduplication in Response to Topoisomerase II Inhibition

The Rad9A checkpoint protein interacts with and is required for proper localization of topoisomerase II-binding protein 1 (TopBP1) in response to DNA damage. Topoisomerase II (Topo II), another binding partner of TopBP1, decatenates sister chromatids that become intertwined during replication. Inhibition of Topo II by ICRF-193 (meso-4,4'-(3,2-butanediyl)-bis-(2,6-piperazinedione)), a catalytic inhibitor that does not induce DNA double-strand breaks, causes a mitotic delay known as the G(2) decatenation checkpoint. Here, we demonstrate that this checkpoint, dependent on ATR and BRCA1, also requires Rad9A. Analysis of different Rad9A phosphorylation mutants suggests that these modifications are required to prevent endoreduplication and to maintain decatenation checkpoint arrest. Furthermore, Rad9A Ser(272) is phosphorylated in response to Topo II inhibition. ICRF-193 treatment also causes phosphorylation of an effector kinase downstream of Rad9A in the DNA damage checkpoint pathway, Chk2, at Thr(68). Both of these sites are major targets of phosphorylation by the ATM kinase, although it has previously been shown that ATM is not required for the decatenation checkpoint. Examination of ataxia telangectasia (A-T) cells demonstrates that ATR does not compensate for ATM loss, suggesting that phosphorylation of Rad9A and Chk2 by ATM plays an additional role in response to Topo II inhibition than checkpoint function alone. Finally, we have shown that murine embryonic stem cells deficient for Rad9A have higher levels of catenated mitotic spreads than wild-type counterparts. Together, these results emphasize the importance of Rad9A in preserving genomic integrity in the presence of catenated chromosomes and all types of DNA aberrations.

Miz1 and HectH9 regulate the stability of the checkpoint protein, TopBP1

The Myc-associated zinc-finger protein, Miz1, activates transcription of the p21cip1 gene in response to UV irradiation. Miz1 associates with topoisomerase II binding protein1 (TopBP1), an essential activator of the Atr kinase. We show here that Miz1 is required for the recruitment of a fraction of TopBP1 to chromatin, for the protection of TopBP1 from proteasomal degradation and for Atr-dependent signal transduction. TopBP1 that is not bound to chromatin is degraded by the HectH9 (Mule, ARF-BP1 and HUWE1) ubiquitin ligase. Myc antagonizes the binding of TopBP1 to Miz1; as a result, expression of Myc leads to dissociation of TopBP1 from chromatin, reduces the amount of total TopBP1 and attenuates Atr-dependent signal transduction. Our data show that Miz1 and Myc affect the activity of the Atr checkpoint through their effect on TopBP1 chromatin association and stability.

Isolation of cross-reacting antigen candidates by mRNA-display using a mixed cDNA library

The identification of cross-reacting antigens is an important process in the characterization of antibodies. We describe here the application of mRNA-display technology with a cDNA library for the isolation of cross-reacting antigens using a monoclonal antibody against human tumor protein p53 (hTP53) as a model. A mixed cDNA library constructed from mRNAs prepared from several human tissues and cell-lines was used for the mRNA-display. After several rounds of panning, five annotated polypeptides, topoisomeraseII-binding protein 1 (TOBP1), RAS protein activator like 2 isoform 1 (RASAL2), endosome-associated FYVE-domain protein (ZFYVE16), and utrophin (UTRN) as well as hTP53, were identified as cross-reactive antigen candidates. All of them had a consensus motif, -S-D-L-( )-K-L-, which was included in the known epitope of the antibody. They were synthesized in vitro, and their binding was compared by conducting a pull-down assay. In cross-activity to the antibody, they ranked as follows: ZYVE16 congruent with hTP53 congruent with TOBP1 > UTRN > RASAL2.

Reconstitution of a human ATR-mediated checkpoint response to damaged DNA

The DNA damage checkpoint response delays cell cycle progression upon DNA damage and prevents genomic instability. Genetic analysis has identified sensor, mediator, signal transducer, and effector components of this global signal transduction pathway. Here we describe an in vitro system with purified human checkpoint proteins that recapitulates key elements of the DNA damage checkpoint. We show that the damage sensor ATR in the presence of topoisomerase II binding protein 1 (TopBP1) mediator/adaptor protein phosphorylates the Chk1 signal-transducing kinase in a reaction that is strongly dependent on the presence of DNA containing bulky base lesions. The dependence on damaged DNA requires DNA binding by TopBP1, and, indeed, TopBP1 shows preferential binding to damaged DNA. This in vitro system provides a useful platform for mechanistic studies of the human DNA damage checkpoint response.

Increasing oxidative damage and loss of mismatch repair enzymes during breast carcinogenesis

This study examined the expression of oxidative damage markers 8-hydroxydeoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (HNE) and nitrotyrosine using immunohistochemical techniques. In addition, DNA topoisomerase II binding protein 1 (TopBP1) and mismatch repair proteins 2 and 6 (MSH2 and MSH6) were immunostained in a series of 80 stage I invasive breast tumours, 26 in situ breast carcinomas and 12 benign breast hyperplasias. 8-OHdG, HNE and nitrotyrosine expression were considerably weaker in hyperplasias than in in situ lesions, which, in turn, showed less oxidative damage than T1N0 tumours. Hyperplasias and in situ tumours were all, at least moderately, positive for MSH2, and nearly all were positive for MSH6. Nitrotyrosine expression was associated with HNE ( P < 0.0005) and 8-OHdG ( P = 0.041) in the T1N0 cohort. To conclude, there is increasing oxidative stress during the early steps of breast carcinogenesis. On the other hand, a significant reduction in expression of mismatch repair proteins occurs during the progression of in situ lesions to invasive tumours.

Human TopBP1 Ensures Genome Integrity during Normal S Phase

Cell cycle checkpoints are essential for maintaining genomic integrity. Human topoisomerase II binding protein 1 (TopBP1) shares sequence similarity with budding yeast Dpb11, fission yeast Rad4/Cut5, and Xenopus Cut5, all of which are required for DNA replication and cell cycle checkpoints. Indeed, we have shown that human TopBP1 participates in the activation of replication checkpoint and DNA damage checkpoints, following hydroxyurea treatment and ionizing radiation. In this study, we address the physiological function of TopBP1 in S phase by using small interfering RNA. In the absence of exogenous DNA damage, TopBP1 is recruited to replicating chromatin. However, TopBP1 does not appear to be essential for DNA replication. TopBP1-deficient cells have increased H2AX phosphorylation and ATM-Chk 2 activation, suggesting the accumulation of DNA double-strand breaks in the absence of TopBP1. This leads to formation of gaps and breaks at fragile sites, 4N accumulation, and aberrant cell division. We propose that the cellular function of TopBP1 is to monitor ongoing DNA replication. By ensuring proper DNA replication, TopBP1 plays a critical role in the maintenance of genomic stability during normal S phase as well as following genotoxic stress.

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.