The BRCT Regions of Tumor Suppressor BRCA1 and of XRCC1 Show DNA End Binding Activity with a Multimerizing Feature

doi:10.1006/bbrc.2000.3983

Biochemical and Biophysical Research Communications

Volume 279, Issue 2, 20 December 2000, Pages 678-684

https://doi.org/10.1006/bbrc.2000.3983 Get rights and content

Abstract

The BRCT regions are two repeating structures in $BR$ CA1 at the $c$ arboxyl- $t$ erminus and are ubiquitous in some proteins involved in cell cycle checkpoint and in DNA repair. Here, using electron microscopy, we show direct evidence that the BRCT regions of BRCA1 bound double-strand breaks of DNA. The BRCT regions could multimerize thus forming large protein particles. Smeared patterns of DNA fragments were consistently shown in the gel retardation assay. A single BRCT was sufficient for DNA binding. The smeared patterns were also observed in BRCTs of TopBP1, suggesting that multimerization may be an important feature of BRCTs. The recombinant second BRCT of XRCC1 (X-ray repair cross-complementing group 1), whose folding was determined by X-ray crystallography, also showed similar DNA end binding images. It is possible that some BRCTs are fundamental structures that detect DNA damages.

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Cited by (48)

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2020, Prognostic and Therapeutic Applications of RKIP in Cancer
Proto-oncogenes and tumor suppressors mediate the complex molecular signaling web involved in the maintenance and control of cell growth, proliferation, and epithelial-to-mesenchymal transition, and disruptions in these pathways can often lead to cancer. To date, several tumor suppressor gene products have been characterized and their dysregulations have been associated in the pathogenesis of various human cancers. More than one tumor suppressor gene product may be expressed in the same cell, and their activities may be independent or overlapping. In addition, the regulation of tumor suppressors' expressions may be the result of crosstalks among several signaling pathways. We, therefore, have initiated a study to determine the presence or absence of a regulatory network via crosstalks of two tumor suppressor gene products expressed in normal human breast tissues and human breast carcinomas: the Raf-1 kinase inhibitor protein (RKIP) and the Breast Cancer Type 1 susceptibility protein (BRCA1). Based on their underlying common functions, we have hypothesized that the expression of these two tumor suppressors is regulated by various crosstalk-mediated signaling pathways. RKIP is a metastasis suppressor frequently expressed in normal tissues, but absent in many human cancers; it has been reported to be involved in the regulation of tumor cell proliferation and viability, invasion and metastasis, and resistance to chemo-immuno therapeutic drugs. BRCA1 is a tumor suppressor protein involved in the repair of damaged DNA, and it is frequently mutated or under-expressed in breast and ovarian cancers. To test our hypothesis, we have analyzed the reported scientific literature to extrapolate the coordinate regulatory and effector mechanisms shared between RKIP and BRCA1. Noteworthy, a broad library of studies has been published on these two proteins individually, but at present there were no reports about the potential interactions that may co-exist between the pathways that regulate RKIP and BRCA1 expressions. Interestingly, our research analyses revealed the presence of five pathways that established each crosstalk that regulate RKIP and BRCA1 expressions. These pathways with crosstalks consist of (1) signaling modules involving p53, (2) regulations of Snail and Slug, (3) beta-catenin expression, (4) Myc regulation, and (5) activation of JAK/STAT. Our delineation of these crosstalks suggested that some tumor suppressor functions in the tumor cell may be interdependent and, further, indicated the potential therapeutic targeting of one crosstalk that will also affect other related tumor suppressor gene products, thus, consequently, resulting in the enhancement of the therapeutic intervention. Our findings herein also point out to the potential of new areas of research investigations to better understand the ontogeny of tumor suppressors, their regulation in cancers, and their therapeutic implications.
The structural basis of XRCC1-mediated DNA repair
2015, DNA Repair
Citation Excerpt :
This suggests that Lig3α may be the preferred ligating enzyme in repairs involving modified DNA termini that require these additional factors as part of the repair process. There are multiple reports of the interaction of XRCC1 and its component domains with DNA [14,23,47,106,111–115], however no structural data for the corresponding DNA complexes is available. A common finding is a lack of significant affinity for double-stranded DNA (dsDNA), but relatively high affinity for nicked, gapped, or blunt-ended DNA.
Scaffold proteins play a central role in DNA repair by recruiting and organizing sets of enzymes required to perform multi-step repair processes. X-ray cross complementing group 1 protein (XRCC1) forms enzyme complexes optimized for single-strand break repair, but participates in other repair pathways as well. Available structural data for XRCC1 interactions is summarized and evaluated in terms of its proposed roles in DNA repair. Mutational approaches related to the abrogation of specific XRCC1 interactions are also discussed. Although substantial progress has been made in elucidating the structural basis for XRCC1 function, the molecular mechanisms of XRCC1 recruitment related to several proposed roles of the XRCC1 DNA repair complex remain undetermined.
Phosphopeptide interactions with BRCA1 BRCT domains: More than just a motif
2015, Progress in Biophysics and Molecular Biology
BRCA1 BRCT domains function as phosphoprotein-binding modules for recognition of the phosphorylated protein-sequence motif pSXXF. While the motif interaction interface provides strong anchor points for binding, protein regions outside the motif have recently been found to be important for binding affinity. In this review, we compare the available structural data for BRCA1 BRCT domains in complex with phosphopeptides in order to gain a more complete understanding of the interaction between phosphopeptides and BRCA1-BRCT domains.
Yeast DNA ligase IV mutations reveal a nonhomologous end joining function of BRCT1 distinct from XRCC4/Lif1 binding
2014, DNA Repair
LIG4/Dnl4 is the DNA ligase that (re)joins DNA double-strand breaks (DSBs) via nonhomologous end joining (NHEJ), an activity supported by binding of its tandem BRCT domains to the ligase accessory protein XRCC4/Lif1. We screened a panel of 88 distinct ligase mutants to explore the structure–function relationships of the yeast Dnl4 BRCT domains and inter-BRCT linker in NHEJ. Screen results suggested two distinct classes of BRCT mutations with differential effects on Lif1 interaction as compared to NHEJ completion. Validated constructs confirmed that D800K and GG(868:869)AA mutations, which target the Lif1 binding interface, showed a severely defective Dnl4–Lif1 interaction but a less consistent and often small decrease in NHEJ activity in some assays, as well as nearly normal levels of Dnl4 accumulation at DSBs. In contrast, mutants K742A and KTT(742:744)ATA, which target the β3-α2 region of the first BRCT domain, substantially decreased NHEJ function commensurate with a large defect in Dnl4 recruitment to DSBs, despite a comparatively greater preservation of the Lif1 interaction. Together, these separation-of-function mutants indicate that Dnl4 BRCT1 supports DSB recruitment and NHEJ in a manner distinct from Lif1 binding and reveal a complexity of Dnl4 BRCT domain functions in support of stable DSB association.
Human Rap1 interacts directly with telomeric DNA and regulates TRF2 localization at the telomere
2012, Journal of Biological Chemistry
Citation Excerpt :
Similarly, an 18-amino acid stretch with five positive charges is present at the N-terminal BRCT domain of hRap1 (10). This is consistent with the finding of a single BRCT domain involved in the DNA binding of TopBP1 (42), XRCC1 (43) and replication factor C (44) to ds-ss junctions. Thus, the BRCT domain of hRap1 may account for its binding to these ds-ss junctions.
The TRF2-Rap1 complex suppresses non-homologous end joining and interacts with DNAPK-C to prevent end joining. We previously demonstrated that hTRF2 is a double strand telomere binding protein that forms t-loops in vitro and recognizes three- and four-way junctions independent of DNA sequence. How the DNA binding characteristics of hTRF2 to DNA is altered in the presence of hRap1 however is not known. Here we utilized EM and quantitative gel retardation to characterize the DNA binding properties of hRap1 and the TRF2-Rap1 complex. Both gel filtration chromatography and mass analysis from two-dimensional projections showed that the TRF2-Rap1 complex exists in solution and binds to DNA as a complex consisting of four monomers each of hRap1 and hTRF2. EM revealed for the first time that hRap1 binds to DNA templates in the absence of hTRF2 with a preference for double strand-single strand junctions in a sequence independent manner. When hTRF2 and hRap1 are in a complex, its affinity for ds telomeric sequences is 2-fold higher than TRF2 alone and more than 10-fold higher for telomeric 3′ ends. This suggests that as hTRF2 recruits hRap1 to telomeric sequences, hRap1 alters the affinity of hTRF2 and its binding preference on telomeric DNA. Moreover, the TRF2-Rap1 complex has higher ability to re-model telomeric DNA than either component alone. This finding underlies the importance of complex formation between hRap1 and hTRF2 for telomere function and end protection.
Structure-function of the tumor suppressor BRCA1
2012, Computational and Structural Biotechnology Journal
Citation Excerpt :
Binding partners for the BRCA1 BRCT domain include BACH1, CtIP, and CCDC98/abraxas [57–59]. While the main function of the BRCA1 BRCT domain is modulating interactions between BRCA1 and phosphoproteins, BRCT domains, including the BRCT domain of BRCA1, can also mediate DNA binding and non-phosphoprotein interactions [60]. Amino acids 1650–1863 of BRCA1 consist of two tandem BRCT repeats connected by a 22 amino acid linker [14].
BRCA1, a multi-domain protein, is mutated in a large percentage of hereditary breast and ovarian cancers. BRCA1 is most often mutated in three domains or regions: the N-terminal RING domain, exons 11–13, and the BRCT domain. The BRCA1 RING domain is responsible for the E3 ubiquitin ligase activity of BRCA1 and mediates interactions between BRCA1 and other proteins. BRCA1 ubiquitinates several proteins with various functions. The BRCA1 BRCT domain binds to phosphoproteins with specific sequences recognized by both BRCA1 and ATM/ATR kinases. Structural studies of the RING and BRCT domains have revealed the molecular basis by which cancer causing mutations impact the functions of BRCA1. While no structural data is available for the amino acids encoded by exons 11–13, multiple binding sites and functional domains exist in this region. Many mutations in exons 11–13 have deleterious effects on the function of these domains. In this mini-review, we examine the structure-function relationships of the BRCA1 protein and the relevance to cancer progression.

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^☆: Abbreviations used: BRCT, BRCA1 carboxyl-terminus; XRCC1, X-ray repair cross-complementing group 1; bp, base pairs; GST, glutathione S-transferase; TBE, 100 mM Tris–borate, 2 mM ethylenediamine tetraacetic acid, pH 8.3.

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Regular ArticleThe BRCT Regions of Tumor Suppressor BRCA1 and of XRCC1 Show DNA End Binding Activity with a Multimerizing Feature☆

Abstract

FEBS Lett.

Gene

Cell

J. Ultrastruct. Res.

J. Mol. Biol.

Dev. Biol.

J. Biol. Chem.

Curr. Biol.

Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs: A cautionary note

Cancer Res.

ASK1 mediates apoptotic cell death induced by genotoxic stress

Oncogene

Activation of c-Abl tyrosine kinase requires caspase activation and is not involved in JNK/SAPK activation during apoptosis of human monocytic leukemia U937 cells

Oncogene

BRCA1 protein products, functional motifs

Nat. Genet.

A superfamily of conserved domains in DNA damage-responsive cell cycle checkpoint proteins

FASEB J.

BRCA1 required for transcription-coupled repair of oxidative DNA damage

Science

Regular Article
The BRCT Regions of Tumor Suppressor BRCA1 and of XRCC1 Show DNA End Binding Activity with a Multimerizing Feature☆