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.     

Regulation of the cyclin D3 promoter by E2F1

We have previously demonstrated that ectopic expression of E2F1 is sufficient to drive quiescent cells into S phase and that E2F1 expression can contribute to oncogenic transformation. Key target genes in this process include master regulators of the cell cycle, such as cyclin E, which regulates G(1) progression, and cyclin A, which is required for the initiation of DNA synthesis. In the present work, we present novel evidence that a second G(1) cyclin, cyclin D3, is also potently activated by E2F1. First, an estrogen receptor-E2F1 fusion protein (ER-E2F1) potently activates the endogenous cyclin D3 mRNA upon treatment with 4-hydroxytamoxifen, which induces nuclear accumulation of the otherwise cytosolic fusion protein. Furthermore, trans-activation of cyclin D3 by ER-E2F1 occurs even in the presence of the protein synthesis inhibitor cycloheximide and thus appears direct. Second, all of the growth-stimulatory members of the E2F family (E2F1, -2, and -3A) potently activate a cyclin D3 promoter reporter, whereas growth-restraining members of the family (E2F4, -5, and -6) have little effect. Third, recombinant E2F1 binds with high affinity to the cyclin D3 promoter in vitro. Fourth, chromatin immunoprecipitation assays demonstrate that endogenous E2F1 is associated with the cyclin D3 promoter in vivo. Finally, mapping experiments localize the essential E2F regulatory element of the cyclin D3 promoter to a noncanonical E2F site in the promoter between nucleotides -143 and -135 relative to the initiating methionine codon. We conclude that in addition to cyclins E and A, E2F family members can also activate one member of the D-type cyclins, further contributing to the ability of the stimulatory E2F family members to drive cellular proliferation.     

Regulation in S Phase by E2F

The DNA synthetic S phase of the unperturbed cell cycle is a closed system, as compared to regulation of G1 by external growth factors. The E2F family provides internal control in S phase by transcribing genes required for deoxynucleotide triphosphate (dNTP) and DNA synthesis. Furthermore, over expression of E2F-1 activates programmed cell death (apoptosis), a safeguarding signal of aberrant growth of cells that have become carcinogenic. Mechanisms for control of E2F-1 are thus essential. The hypothesis is proposed that deoxythymidine triphosphate (dTTP) allosterically feedback controls E2F-1 to regulate both DNA synthesis and apoptosis. This may act either upon production of E2F-1 or its degradation.     

Regulation of isocitrate lyase inRhodobacter capsulatus E1F1

InRhodobacter capsulatus E1F1, isocitrate lyase (ICL) (EC 4.5.3.1) is a regulatory enzyme whose levels are increased in the presence of acetate as the sole carbon source. Acetate activated isocitrate lyase in a process dependent on energy supply and de novo protein synthesis. In contrast to isocitrate lyase, isocitrate dehydrogenase (ICDH) activity was independent of the carbon source used for growth and significantly increased in darkened cells. Pyruvate or yeast extract prevented in vivo activation of isocitrate lyase in cells growing on acetate. The enzyme was reversibly inactivated to a great extent in vitro by pyruvate and other oxoacids presumably involved in acetate metabolism. These results suggest that, inR. capsulatus E1F1, isocitrate lyase is regulated by both enzyme synthesis and oxoacid inactivation.     

Regulation of nitrate reductase inRhodobacter capsulatus E1F1

The photosynthetic purple non-sulfur nitrate-assimilating bacteriumRhodobacter capsulatus E1F1 has an adaptive nitrate reductase activity inducible by either nitrate or nitrite and molybdenum traces. Nitrate reductase induction by nitrate did not occur in media with nitrate and ammonium, which showed no effect if nitrite was the inductor instead of nitrate or in the presence ofl-methionine-dl-sulfoximine (MSX) plus nitrate. In vivo, tungstate inhibited nitrate reductase activity, and this was not recovered upon addition of molybdenum unless de novo protein synthesis took place. Nitrate reductase was also repressed in nitrogen-starved cells or after the addition of azaserine to cells growing phototrophically with nitrate. Moreover, higher rates of nitrate reductase induction and nitrite excretion were found in illuminated cells grown with nitrate under air than in those grown under argon.