Expression of TopBP1 in hereditary breast cancer

TopBP1 protein displays structural as well as functional similarities to BRCA1 and is involved in DNA replication, DNA damage checkpoint response and transcriptional regulation. Aberrant expression of TopBP1 may lead to genomic instability and can have pathological consequences. In this study we aimed to investigate expression of TopBP1 gene at mRNA and protein level in hereditary breast cancer. Real-time quantitative PCR was performed in 127 breast cancer samples. Expression of TopBP1 mRNA in lobular carcinoma was significantly lower compared with ductal carcinoma (p < 0.05). The level of TopBP1 mRNA appeared to be lower in poorly differentiated (III grade) hereditary breast cancer in comparison with moderately (II grade) and well-differentiated cancer (I grade) (p < 0.05 and p < 0.001 respectively). We analyzed TopBP1 protein expression using immunohistochemistry and Western blot techniques. Expression of TopBP1 protein was found to be significantly increased in poorly differentiated breast cancer (III grade) (p < 0.05). The percentage of samples with cytoplasmic apart from nuclear staining increased with increasing histological grade. There was no significant association between level and intracellular localization of TopBP1 protein in hereditary breast cancer and other clinicopathological parameters such as estrogen and progesterone receptors status, appearance of metastasis in the axillary lymph nodes and type of cancer. Our data suggest that decreased level of TopBP1 mRNA and increased level of TopBP1 protein might be associated with progression of hereditary breast cancer

Rad17 Plays a Central Role in Establishment of the Interaction between TopBP1 and the Rad9-Hus1-Rad1 Complex at Stalled Replication Forks

Rad17 is critical for the ATR-dependent activation of Chk1 during checkpoint responses. It is known that Rad17 loads the Rad9-Hus1-Rad1 (9-1-1) complex onto DNA. We show that Rad17 also mediates the interaction of 9-1-1 with the ATR-activating protein TopBP1 in Xenopus egg extracts. Studies with Rad17 mutants indicate that binding of ATP to Rad17 is essential for the association of 9-1-1 and TopBP1. Furthermore, hydrolysis of ATP by Rad17 is necessary for the loading of 9-1-1 onto DNA and the elevated, checkpoint-dependent accumulation of TopBP1 on chromatin. Significantly, a mutant 9-1-1 complex that cannot bind TopBP1 has a normal capacity to promote elevated accumulation of TopBP1 on chromatin. Taken together, we propose the following mechanism. First, Rad17 loads 9-1-1 onto DNA. Second, TopBP1 accumulates on chromatin in a manner that depends on both Rad17 and 9-1-1. Finally, 9-1-1 and TopBP1 dock in a Rad17-dependent manner before activation of Chk1

Consensus over Random Graph Processes: Network Borel-Cantelli Lemmas for Almost Sure Convergence

Distributed consensus computation over random graph processes is considered. The random graph process is defined as a sequence of random variables which take values from the set of all possible digraphs over the node set. At each time step, every node updates its state based on a Bernoulli trial, independent in time and among different nodes: either averaging among the neighbor set generated by the random graph, or sticking with its current state. Connectivity-independence and arc-independence are introduced to capture the fundamental influence of the random graphs on the consensus convergence. Necessary and/or sufficient conditions are presented on the success probabilities of the Bernoulli trials for the network to reach a global almost sure consensus, with some sharp threshold established revealing a consensus zero-one law. Convergence rates are established by lower and upper bounds of the $\epsilon$-computation time. We also generalize the concepts of connectivity/arc independence to their analogues from the $*$-mixing point of view, so that our results apply to a very wide class of graphical models, including the majority of random graph models in the literature, e.g., Erd\H{o}s-R\'{e}nyi, gossiping, and Markovian random graphs. We show that under $*$-mixing, our convergence analysis continues to hold and the corresponding almost sure consensus conditions are established. Finally, we further investigate almost sure finite-time convergence of random gossiping algorithms, and prove that the Bernoulli trials play a key role in ensuring finite-time convergence. These results add to the understanding of the interplay between random graphs, random computations, and convergence probability for distributed information processing.Comment: IEEE Transactions on Information Theory, In Pres

Rad4(TopBP1), a Scaffold Protein, Plays Separate Roles in DNA Damage and Replication Checkpoints and DNA Replication

Rad4(TopBP1), a BRCT domain protein, is required for both DNA replication and checkpoint responses. Little is known about how the multiple roles of Rad4(TopBP1) are coordinated in maintaining genome integrity. We show here that Rad4(TopBP1) of fission yeast physically interacts with the checkpoint sensor proteins, the replicative DNA polymerases, and a WD-repeat protein, Crb3. We identified four novel mutants to investigate how Rad4(TopBP1) could have multiple roles in maintaining genomic integrity. A novel mutation in the third BRCT domain of rad4(+TopBP1) abolishes DNA damage checkpoint response, but not DNA replication, replication checkpoint, and cell cycle progression. This mutant protein is able to associate with all three replicative polymerases and checkpoint proteins Rad3(ATR)-Rad26(ATRIP), Hus1, Rad9, and Rad17 but has a compromised association with Crb3. Furthermore, the damaged-induced Rad9 phosphorylation is significantly reduced in this rad4(TopBP1) mutant. Genetic and biochemical analyses suggest that Crb3 has a role in the maintenance of DNA damage checkpoint and influences the Rad4(TopBP1) damage checkpoint function. Taken together, our data suggest that Rad4(TopBP1) provides a scaffold to a large complex containing checkpoint and replication proteins thereby separately enforcing checkpoint responses to DNA damage and replication perturbations during the cell cycle

An interaction between human papillomavirus 16 E2 and TopBP1is required for optimum viral DNA replication and episomal genome establishment

In human papillomavirus DNA replication the viral protein E2 forms homodimers and binds to 12bp palindromic DNA sequences surrounding the origin of DNA replication. Via a protein-protein interaction it then recruits the viral helicase E1 to an A/T rich origin of replication whereupon a di-hexamer forms resulting in DNA replication initiation. In order to carry out DNA replication the viral proteins must interact with host factors that are currently not all known. An attractive cellular candidate for regulating viral replication is TopBP1, a known interactor of the E2 protein. In mammalian DNA replication TopBP1 loads DNA polymerases onto the replicative helicase following the G1 to S transition and this process is tightly cell cycle controlled. The direct interaction between E2 and TopBP1 would allow E2 to by-pass this cell cycle control resulting in DNA replication more than once per cell cycle which is a requirement for the viral life cycle. Here we report the generation of an HPV16 E2 mutant compromised in TopBP1 interaction in vivo and demonstrate that this mutant retains transcriptional activation and repression functions but has sub-optimal DNA replication potential. Introduction of this mutant into a viral life cycle model results in the failure to establish viral episomes. The results present a potential new anti-viral target, the E2-TopBP1 interaction, and increase our understanding of the viral life cycle suggesting that the E2-TopBP1 interaction is essential