The genomic integrity of a cell is constantly being pressured by both intrinsic and extrinsic forces. Cell cycle checkpoints exist to protect the cells by arresting cell cycle progression in response to DNA damage or replication stress. It has been shown that the interaction between the checkpoint proteins Rad9A and TopBP1 is a crucial upstream event required for the ATR-dependent checkpoint response to DNA damage, which can be activated throughout different points in the cell cycle. The Bimolecular Fluorescence Complementation (BiFC) technique has recently emerged as a simple and effective tool for analyzing protein-protein interactions in live cell cultures. By fusing complementary fragments of fluorescent proteins to proteins of interest, one can visualize protein-protein interactions through the formation of a mature fluorophore from these fragments. In the current work, the BiFC assay system was employed to study the interaction between TopBP1 and Rad9A; the human homologue of fission yeast Rad9. BiFC vectors expressing TopBP1, Rad9A, and the Rad9A-S387 mutant were constructed and optimized for transfection in HeLa cells. It was shown that the BiFC fusion protein of Rad9A lacked phosphorylation on its constitutive S387 site, although it retained its upstream damage dependent S272 phosphorylation after IR treatment. BiFC signals could be detected in cells containing the BiFC fusion proteins of Rad9A and TopBP1 using confocal microscopy and flow cytometry techniques. However, the signals could not be distinguished from that of the negative control samples. Our results suggest a possibility that our BiFC fusion proteins of interest interact in a non-specific manner, although further characterization is required to confirm this. The BiFC assay employed in this project must be further optimized to effectively study the interaction between Rad9A and TopBP1, as well as other checkpoint proteins. However, this study has given us great insight into the implementation of this new BiFC technique for studying protein interactions in the context of cell cycle proteins, and the knowledge gained from this study will be invaluable for future work. / Thesis (Master, Biochemistry) -- Queen's University, 2009-12-17 12:42:06.717

Abstract The human and mouse genes POLE1 and Pole1 for the catalytic subunit of DNA polymerase ε contain 51 and 49 exons, respectively, and the human gene POLE2 for the B-subunit contains 19 exons. The human POLE1 encodes three alternatively spliced mRNAs differing in their 5'-terminal sequence and in the N-termini of the predicted proteins. The promoters for the major human transcript and the mouse Pole1 are G+C rich, TATA-less and contain putative cis-acting elements typical of both S phase upregulated and serum responsive promoters. Interestingly, the three human alternative transcripts are expressed from three promoters, and other structural features of POLE1 suggest that regulation of its expression is complicated. The amino acid sequence of the catalytic subunit deduced from the mouse cDNA shows remarkable evolutionary conservation in the DNA polymerase ε family. Interestingly, several conserved elements involved in template-primer binding differ from those of other class B DNA polymerases. This is likely to reflect a distinctive function of the enzyme. The mouse Pole1 was localized to chromosome 5 region E3-E5. The expression of the human POLE2 encoding the B-subunit of DNA polymerase ε is dependent on cell proliferation in a late serum-responsive manner. This is typical for DNA replication-related proteins. The promoter, which utilizes multiple transcriptional initiation sites, is G+C rich and lacks a TATA-box. A 75 bp core promoter region is located within exon 1 and contains an Sp1 element as a critical determinant of the promoter activity. Two overlapping E2F elements adjacent to the Sp1 element are essential for full promoter activity and serum response. Immediately downstream from the core promoter region reside binding sites for E2F1 and NF-1. POLE2 seems to be regulated by two E2F-pocket protein complexes, one associated with Sp1 and the other with NF-1. The complete cDNA of the human DNA topoisomerase IIβ binding protein (TopBP1) reveals a 170 kDa protein that contains eight BRCT-domains and shows homology to S. cerevisiae Dpb11 and S. pombe Cut5/Rad4. The protein interacts physically with human DNA polymerase ε as shown by co-immunoprecipitation. A peptide containing the 6th BRCT-domain and an antibody against this peptide inhibit DNA replication in isolated nuclei, indicating that the protein is required for DNA replication. The expression of TopBP1 is proliferation-dependent in a manner that is typical for replication proteins. The gene encoding TopBP1 was localized to chromosome 3q21-q23.

No description available.

Abstract The currently known susceptibility genes account for approximately 25% of familial breast cancer predisposition. Additional factors contributing to the pathogenesis of breast cancer are, therefore, likely to be discovered. Most of the known genes affecting breast cancer predisposition function in the DNA damage response pathway. In this study three genes, TOPBP1, CLSPN and PALB2, involved in this complex process were investigated to reveal potentially pathogenic mutations associated with breast cancer susceptibility. In the analysis of the TOPBP1 gene, one novel putative pathogenic alteration was observed. The Arg309Cys variant was found at an elevated frequency among familial cases (19/125) vs. controls (49/697) (p = 0.002; OR 2.4; 95% CI 1.3–4.2). In addition, altogether 18 other germline alterations were observed in this gene, but they all appeared to be harmless polymorphisms. Investigation of CLSPN alterations among familial breast cancer families revealed altogether seven different changes. No clearly pathogenic alterations were observed. However, a potential modifier effect was discovered for the 1195delGlu change. The obtained results suggest that CLSPN alterations are unlikely to be significant breast cancer susceptibility alleles. In the PALB2 gene, a pathogenic mutation c.1592delT was identified at an elevated frequency among breast cancer patients (0.9%) compared to controls (0.2%) (p = 0.003, OR 3.94, 95% CI 1.5–12.1). Among familial cases the frequency of c.1592delT was even higher (2.7%). This mutation was also functionally deficient. It had a markedly decreased BRCA2-binding affinity and was unable to support homologous recombination or to restore cross link repair in PALB2 knock-down cells. Additionally, this mutation was discovered in a familial prostate cancer family and was found to segregate with the disease, suggesting some association also with prostate cancer. The penetrance and hazard ratio associated with PALB2 c.1592delT were determined in unselected breast cancer families. A substantially increased risk of breast cancer (HR 6.1; 95% CI 2.2–17.2; p = 0.01) was discovered resulting in an estimated 40% (95% CI 17–77) breast cancer risk by age 70 years, comparable to that for carriers of BRCA2 mutations. This markedly increased cancer risk suggests that genetic counselling for carriers is needed and screening for this mutation should be considered.

Abstract Genetic information is stored in the base sequence of DNA. As DNA is often damaged by radiation or reactive chemicals, cells have developed mechanisms to correct the DNA lesions. These mechanisms involve recognition of damage, DNA repair and cell cycle delay until DNA is restored. Failures in the proper processing of DNA lesions may lead to mutations, premature aging, or diseases such as cancer. In this thesis study the human topoisomerase IIβ binding protein 1 (TopBP1) was identified as the homolog of budding yeast Dpb11 and fission yeast Cut5. TopBP1 was found to be necessary for DNA replication and to associate with replicative DNA polymerase ε. TopBP1 localised to the sites of DNA damage and stalled replication forks, which suggests a role in the DNA damage response. TopBP1 interacted with the checkpoint protein Rad9, which is a part of a protein complex whose function includes tethering proteins to sites of DNA damage. This supports a role for TopBP1 in the early steps of checkpoint activation after DNA damage. TopBP1 also interacted with the tumour suppressor protein p53 in a phosphorylation dependent manner. In addition, the data support a role for TopBP1 outside of S-phase. During M-phase, TopBP1 was found to localise to centrosomes along with the tumour suppressor proteins Brca1 and p53. Analysis of the expression of TopBP1 in mouse tissues suggested that TopBP1 may also play a role during meiosis. The localisation pattern of TopBP1 in mouse meiotic spermatocytes resembled that of many proteins functioning during meiotic recombination. For example, co-localisation of ATR kinase and TopBP1 was observed during meiotic prophase I. In accordance with the findings from mouse studies, the analysis of a cut5 mutant during yeast meiosis showed that Cut5 is essential for the meiotic checkpoint. These results strongly suggest that TopBP1 operates in replication and has checkpoint functions during both the mitotic and meiotic cell cycles.

Abstract DNA polymerase ε from HeLa cells has been purified as a heterodimer of a 261 kDa catalytic subunit and a tightly associated smaller polypeptide, the B-subunit. The cDNAs encoding the B-subunits of both human and mouse Pol ε were cloned and shown to encode proteins with a predicted molecular weight of 59 kDa. These subunits are 90 % identical and share 22 % identity with the 80 kDa B-subunit of Saccharomyces cerevisiae Pol ε. The gene for the human Pol ε B-subunit was localized to chromosome 14q21-q22 by fluorescence in situ hybridization. Primary structure analysis of the Pol ε B-subunits demonstrated that they are similar to the B-subunits of Pol α, Pol δ and archaeal DNA polymerases, and comprise a novel protein family of DNA polymerase associated-B-subunits. The family members have 12 conserved motifs distributed in the C-terminal parts, which apparently form crucial structural and functional sites. Secondary structure predictions indicate that the B-subunits share a similar fold, and phylogenetic analysis demonstrated that the B-subunits of Pol α and ε form one subfamily, while the B-subunits of Pol δ and the archaeal proteins form a second subfamily. The corresponding eukaryotic and archaeal catalytic subunits are not related, but all have the characteristics of replicative DNA polymerases. This indicates that the B-subunits of replicative DNA polymerases from archaea to eukaryotes belong to the same protein family and perform similar functions. In S. cerevisiae, Pol ε associates with the checkpoint protein Dpb11. In this study, a human protein, TopBP1, with structural similarity to the budding yeast Dpb11, fission yeast Cut5 and the breast cancer susceptibility gene product Brca1 was identified. The human TOPBP1 gene localizes to chromosome 3q21-q23 and encodes a phosphoprotein of 180 kDa. TopBP1 has eight BRCT domains and is also closely related to the recently identified Drosophila melanogaster Mus101. TopBP1 expression is induced at the G1/S boundary and it performs an important role in DNA replication, as evidenced by inhibition of DNA synthesis by TopBP1 antiserum in isolated nuclei. TopBP1 also associates with Pol ε and localizes, together with Brca1 to distinct foci in S-phase, but not to sites of ongoing DNA replication. Inhibition of DNA replication leads to re-localization of TopBP1 and Brca1 to stalled replication forks. DNA damage induces formation of distinct TopBP1 foci that co-localize with Brca1 in S-phase, but not in G1-phase. The role of TopBP1 in the DNA damage response is also supported by the interaction between TopBP1 and the human checkpoint protein hRad9. These results implicate TopBP1 in replication and checkpoint functions.

Alternative lengthening of telomeres (ALT) is a telomerase-independent mechanism utilized by a subset of cancers to promote replicative immortality. The ALT mechanism is driven by increased replication stress and persistent DNA damage response signaling that leads to a homology-directed repair mechanism called break-induced telomere synthesis (BITS). In particular, ALT cells are hypersensitive to inhibition of ataxia telangiectasia Rad3-related (ATR), a DNA damage response kinase implicated in telomere mobility and recruitment of repair proteins for telomere elongation in ALT. However, little is known about what regulates ATR activity at ALT telomeres. Given the importance of ATR in the ALT mechanism, we hypothesized that known ATR activators, ETAA1 and TopBP1, regulate ALT activity and telomere synthesis. Here, we show that ETAA1 and TopBP1 localize to ALT telomeres at sites of ALT activity and telomeric damage. Furthermore, depletion of ETAA1 and TopBP1 leads to defects in ATR signaling, a decrease in BITS and compensatory engagement of telomeric MiDAS (spontaneous mitotic telomere synthesis) for replication stress resolution. Taken together, our findings show that both ETAA1 and TopBP1 are important for mediating BITS at ALT telomeres and may better inform our efforts in targeting the ATR signaling pathway in ALT-positive cancers.

No description available.

No description available.

Abstract Approximately 5–10% of all breast cancer cases are estimated to result from a hereditary predisposition to the disease. Currently no more than 25–30% of these familial cases can be explained by mutations in the known susceptibility genes, BRCA1 and BRCA2 being the major ones. Additional predisposing genes are therefore likely to be discovered. This study evaluates whether germline alterations in three BRCA1-associated genes, BACH1 (i.e. BRIP1/FANCJ), BARD1 and TOPBP1, contribute to familial breast cancer. Altogether 214 Finnish patients having breast and/or ovarian cancer were analysed for germline mutations in the BACH1 gene. Nine alterations were observed, four of which located in the protein-encoding region. The previously unidentified Pro1034Leu was considered a possible cancer-associated alteration as it appeared with two-fold higher frequency among cancer cases compared to controls. All the other observed alterations were classified as harmless polymorphisms. Mutation analysis of the BARD1 gene among 126 Finnish patients having family history of breast and/or ovarian cancer revealed seven alterations in the protein-encoding region. The Cys557Ser alteration was seen at an elevated frequency among familial cancer cases compared to controls (p = 0.005, odds ratio [OR] 4.2, 95% confidence interval [CI] 1.7–10.7). The other alterations appeared to be harmless polymorphisms. To evaluate further the possible effect of Cys557Ser on cancer risk, a large case-control study was performed, consisting of 3,956 cancer patients from the Nordic countries. The highest prevalence of Cys557Ser was found among breast and ovarian cancer patients from BRCA1/BRCA2 mutation-negative families (p < 0.001, OR 2.6, 95% CI 1.7–4.0). In contrast, no significant association with male breast cancer, ovarian, colorectal or prostate cancer was observed. The current study is the first evaluating the role of TOPBP1 mutations in familial cancer predisposition. The analysis of 125 Finnish patients having breast and/or ovarian cancer revealed one putative pathogenic alteration. The commonly occurring Arg309Cys allele was observed at a significantly higher frequency among familial cancer cases compared to controls (p = 0.002, OR 2.4, 95% CI 1.3–4.2). The other 18 alterations observed were classified as harmless polymorphisms.