Characterization of the interaction between the human DNA topoisomerase IIβ-binding protein 1 (TopBP1) and the cell division cycle 45 (Cdc45) protein

2007 ◽  
Vol 409 (1) ◽  
pp. 169-177 ◽  
Author(s):  
Uta Schmidt ◽  
Yvonne Wollmann ◽  
Claudia Franke ◽  
Frank Grosse ◽  
Hans-Peter Saluz ◽  
...  
Keyword(s):  
Cell Division ◽  
Dna Replication ◽  
Transcriptional Activation ◽  
Glutathione Transferase ◽  
Cancer Susceptibility ◽  
Binding Protein ◽  
Susceptibility Gene ◽  
Cell Division Cycle ◽  
Breast Cancer Susceptibility Gene

TopBP1 (topoisomerase IIβ-binding protein 1) is a BRCT [BRCA1 (breast-cancer susceptibility gene 1) C-terminal]-domain-rich protein that is structurally and functionally conserved throughout eukaryotic organisms. It is required for the initiation of DNA replication and for DNA repair and DNA damage signalling. Experiments with fission yeast and Xenopus revealed that the TopBP1 homologues of these organisms are required for chromatin loading of the replication protein Cdc45 (cell division cycle 45). To improve our understanding of the physiological functions of human TopBP1, we investigated the interplay between human TopBP1 and Cdc45 proteins in synchronized HeLa-S3 cells. Using GST (glutathione transferase) pull-down and co-immunoprecipitation techniques, we showed a direct interaction between TopBP1 and Cdc45 in vitro and in vivo. The use of deletion mutants in GST pull-down assays identified the first and second as well as the sixth BRCT domains of TopBP1 to be responsible for the functional interaction with Cdc45. Moreover, the interaction between Cdc45 and the first and second BRCT domains of TopBP1 inhibited their transcriptional activation both in yeast and mammalian one-hybrid systems. Both proteins interacted exclusively at the G1/S boundary of cell cycle; only weak interaction could be found at the G2/M boundary. The overexpression of the sixth BRCT domain led to diminished loading of Cdc45 on to chromatin. These results suggest that human TopBP1 is involved in the formation of the initiation complex of replication in human cells and is required for the recruitment of Cdc45 to origins of DNA replication.

scholarly journals Host Cell Reactivation and Transcriptional Activation of Carboplatin-Modified BRCA1

2014 ◽  
Vol 8 ◽  
pp. BCBCR.S14224 ◽  
Author(s):  
Adisorn Ratanaphan ◽  
Bhutorn Canyuk
Keyword(s):  
Host Cell ◽  
Transcriptional Activation ◽  
Cancer Susceptibility ◽  
Susceptibility Gene ◽  
Brca1 Gene ◽  
Breast Cancer Susceptibility ◽  
Breast Cancer Susceptibility Gene ◽  
Cell Reactivation ◽  
Host Cell Reactivation

The breast cancer susceptibility gene 1 ( BRCA1) has been shown to maintain genomic stability through multiple functions in the regulation of DNA damage repair and transcription. Its translated BRCT (BRCA1 C-terminal domain) acts as a strong transcriptional activator. BRCA1 damaged by carboplatin treatment may lead to a loss of such functions. To address the possibility of the BRCA1 gene as a therapeutic target for carboplatin, we investigated the functional consequences of the 3′-terminal region of human BRCA1 following in vitro platination with carboplatin. A reduction in cellular BRCA1 repair of carboplatin-treated plasmid DNA, using a host cell reactivation assay, was dependent on the platination levels on the reporter gene. The transcriptional transactivation activity of the drug-modified BRCA1, assessed using a one-hybrid GAL4 transcriptional assay, was inversely proportional to the carboplatin doses. The data emphasized the potential of the BRCA1 gene to be a target for carboplatin treatment.

Transcriptional activation requires protection of the TATA-binding protein Tbp1 by the ubiquitin-specific protease Ubp3

2010 ◽  
Vol 431 (3) ◽  
pp. 391-402 ◽  
Author(s):  
Boon Shang Chew ◽  
Wee Leng Siew ◽  
Benjamin Xiao ◽  
Norbert Lehming
Keyword(s):  
Mutant Strain ◽  
Transcriptional Activation ◽  
Glutathione Transferase ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Mutant Protein ◽  
Specific Protease ◽  
Ubiquitin Specific Protease

Tbp1, the TATA-binding protein, is essential for transcriptional activation, and Gal4 and Gcn4 are unable to fully activate transcription in a Saccharomyces cerevisiae TBP1E86D mutant strain. In the present study we have shown that the Tbp1E186D mutant protein is proteolytically instable, and we have isolated intragenic and extragenic suppressors of the transcription defects of the TBP1E186D mutant strain. The TBP1R6S mutation stabilizes the Tbp1E186D mutant protein and suppresses the defects of the TBP1E186D mutant strain. Furthermore, we found that the overexpression of the de-ubiquitinating enzyme Ubp3 (ubiquitin-specific protease 3) also stabilizes the Tbp1E186D mutant protein and suppresses of the defects of the TBP1E186D mutant strain. Importantly, the deletion of UBP3 and its cofactor BRE5 lead to increased degradation of wild-type Tbp1 protein and to defects in transcriptional activation by Gal4 and Gcn4. Purified GST (glutathione transferase)–Ubp3 reversed Tbp1 ubiquitination, and the deletion of UBP3 lead to the accumulation of poly-ubiquitinated species of Tbp1 in a proteaseome-deficient genetic background, demonstrating that Ubp3 reverses ubiquitination of Tbp1 in vitro and in vivo. Chromatin immunoprecipitation showed that Ubp3 was recruited to the GAL1 and HIS3 promoters upon the induction of the respective gene, indicating that protection of promoter-bound Tbp1 by Ubp3 is required for transcriptional activation.

DNA damage hypersensitivity in cells lacking BRCA2: a review of in vitro and in vivo data

2005 ◽  
Vol 33 (4) ◽  
pp. 715-717 ◽  
Author(s):  
T. Hay ◽  
A.R. Clarke
Keyword(s):  
Cancer Susceptibility ◽  
Susceptibility Gene ◽  
Short Review ◽  
Breast Cancer Susceptibility ◽  
Breast Cancer Susceptibility Gene ◽  
Functional Protein ◽  
Increased Sensitivity ◽  
Cancer Susceptibility Gene

Since the discovery of the tumour suppressor BRCA2 (encoded by breast-cancer susceptibility gene 2), cells lacking the fully functional protein have consistently been found to show increased sensitivity to a variety of DNA-damaging agents, particularly those that cross-link DNA. In this short review, we will bring together these findings and discuss them in the light of our recent in vivo data in the mouse small intestine, which suggests that deletion of cells lacking Brca2 is necessary to avoid the development of potentially tumorigenic clones in this tissue, a system that may be less effective in the mammary glands of humans with germline mutations in BRCA2.

scholarly journals Dual regulation of AP-2α transcriptional activation by poly(ADP-ribose) polymerase-1

2004 ◽  
Vol 382 (1) ◽  
pp. 323-329 ◽  
Author(s):  
Min LI ◽  
Padmavathy NAIDU ◽  
Yihong YU ◽  
Nathan A. BERGER ◽  
Perry KANNAN
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Catalytic Domain ◽  
Cancer Susceptibility ◽  
Susceptibility Gene ◽  
Enzymic Activity ◽  
Breast Cancer Susceptibility Gene ◽  
Target Sequence ◽  
Middle Region ◽  
Parp 1

Poly(ADP-ribose) polymerase-1 (PARP-1) is a co-activator for AP-2α (activator protein 2α)-mediated transcriptional activation. In the present study, we find that the role of PARP-1 in AP-2α transcription is distinctly dualistic with opposing effects. Separate regions of PARP-1 interact with AP-2α and independently control its transcriptional activation. The C-terminus containing the catalytic domain strongly interacts with AP-2α, whereas low-affinity binding is seen in the middle region, which includes the breast-cancer susceptibility gene 1 C-terminal domain and automodification region. The middle region enhances AP-2α transcription. Even portions of this region independently interact and have partial effects on transcription. The catalytic domain strongly poly-(ADP-ribosyl)ates AP-2α. This modification, on the other hand, affects its DNA binding. 3-Aminobenzamide and 6(5H)-phenanthridinone that inhibit the enzymic activity significantly enhance the binding of AP-2α to its target sequence and increase its transcriptional activity. The enzymic activity of PARP-1 is known to be induced by stress conditions that damage cellular DNA, and the poly(ADP-ribosyl)ation of target proteins is transient in nature with a half-life of less than a minute. We hypothesize that PARP-1 enhances the transcriptional activity of AP-2α in normal circumstances, whereas its enzymic activity is used as a temporary shut-off mechanism during unfavourable conditions.

scholarly journals The breast cancer susceptibility gene, BRCA2 : at the crossroads between DNA replication and recombination?

2000 ◽  
Vol 355 (1394) ◽  
pp. 191-198 ◽  
Author(s):  
Ashok R. Venkitaraman
Keyword(s):  
Breast Cancer ◽  
Dna Replication ◽  
Cancer Susceptibility ◽  
Susceptibility Gene ◽  
Breast Cancer Susceptibility ◽  
Breast Cancer Susceptibility Gene ◽  
Biological Functions ◽  
Mammalian Homologue ◽  
Critical Requirement ◽  
Cancer Susceptibility Gene

The identification and cloning of the familial breast cancer susceptibility gene, BRCA2 , has excited much interest in its biological functions. Here, evidence is reviewed that the protein encoded by BRCA2 has an essential role in DNA repair through its association with mRad51, a mammalian homologue of bacterial and yeast proteins involved in homologous recombination. A model is proposed that the critical requirement for BRACA2 in cell division and the maintenance of chromosome stability stems from its participation in recombinational processes essential for DNA replication.

scholarly journals TopBP1 contains a transcriptional activation domain suppressed by two adjacent BRCT domains

2006 ◽  
Vol 400 (3) ◽  
pp. 573-582 ◽  
Author(s):  
Roni H. G. Wright ◽  
Edward S. Dornan ◽  
Mary M. Donaldson ◽  
Iain M. Morgan
Keyword(s):  
Dna Damage ◽  
Transcriptional Activation ◽  
Cancer Susceptibility ◽  
Susceptibility Gene ◽  
Chromatin Modification ◽  
Breast Cancer Susceptibility Gene ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Regulatory Domains ◽  
Transcriptional Regulatory

TopBP1 has eight BRCT [BRCA1 (breast-cancer susceptibility gene 1) C-terminus] domains and is involved in initiating DNA replication, and DNA damage checkpoint signalling and repair. Several BRCT-domain-containing proteins involved in mediating DNA repair have transcriptional regulatory domains, and as demonstrated for BRCA1 these regulatory domains are important in mediating the functions of these proteins. These transcriptional regulatory processes involve modification of chromatin, and recent evidence has clearly demonstrated that the ability to modify chromatin plays an important role in regulating DNA damage signalling and repair. Here we report the identification of a TopBP1 transcriptional activation domain that is rich in hydrophobic residues, interspersed with acidic amino acids, characteristics that are typical of transcriptional activation domains identified previously. Two adjacent repressor domains encoded by BRCT2 and BRCT5 silence this activator and experiments suggest that these repressors actively recruit repressor complexes. Both the activator and BRCT2 repressor domains function in yeast. The present study identifies several chromatin modification domains encoded by TopBP1, and the implications of these findings are discussed in the context of the DNA damage response and the understanding of TopBP1 function.

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