BRCA1 and BRCT proteins as phophopeptide binding modules in DNA damage signaling
BRCA1 and BRCT proteins as phophopeptide binding modules in DNA damage signaling

The cloning of the first gene associated with hereditary breast cancer, BRCA1, provided an important tool for breast cancer screening, however the predicted amino acid sequence of the protein revealed little about its possible function. Sequence analysis revealed weak repeated sequences at the C-terminus of BRCA1 that were termed the BRCT repeats (BRCA1 C-terminal repeats). This analysis, together with the fact that many of the cancer-associated mutations clustered within this region, suggested that the BRCT region could be critical for BRCA1 function.

Structure of the BRCA1 BRCT domain

We began our work to crystallize this region in 1998, with the idea that the structure might reveal something about the structure. Our structure, published in 2001, revealed that each BRCT repeat adopted a mixed α-β fold and that the tandem BRCT repeats packed in a head-to-tail manner. While we could not predict the function of this region from the structure, we were able to show that cancer-associated missense mutations could disrupt the folding of the BRCT domain by disrupting the head-to-tail packing of the BRCT repeats.

Phosphorylation regulates the DNA damage response
Figure 1. Structure of the BRCA1 BRCT domain.
Williams, R. S., Green, R. and Glover, J. N. M. (2001) Crystal structure of the BRCT repeat region from the breast cancer-associated protein BRCA1. Nature Structural Biology 8(10):838-842. PDF
The BRCA1 BRCT repeats act as a phospho-peptide binding module

Protein phosphorylation critically regulates protein-protein signaling interactions that govern the DNA damage response. Work from Junjie Chen and Mike Yaffe showed that the BRCA1 BRCT domains act to recognize specific phosphorylated motifs in BRCA1 partner proteins such as the BACH1 DNA helicase. We uncovered the structural basis for BRCA1 recognition of its target pSer-x-x-Phe peptide motif. Our work revealed a pSer binding pocket in the N-terminal BRCT repeat, and a secondary, Phe-binding pocket at the interface between the two repeats. Cancer associated mutations specifically blocked the peptide binding site, and we could predict that a number of other BRCT proteins might also have phospho-peptide binding activity.

Phosphorylation regulates the DNA damage response
Figure 2. The BRCA1 BRCT repeats act as a phospho-peptide binding module
Williams, R. S., Lee, M. S., Hau, D. D. and Glover, J. N. M. (2004) Structural basis of phosphopeptide recognition by the BRCT domain of BRCA1. Nature Structural and Molecular Biology 11(6):519-525. PDF
Glover, J. N. M., Williams, R. S. and Lee, M. S. (2004) Interactions between BRCT repeats and phosphoproteins: Tangled up in two. Trends in Biochemistry Science 29(11):579-585. (Review Article) PDF
Campbell, S. J., Edwards, R. A. and Glover, J. N. M. (2010) Comparison of the structures and peptide binding specificities of the BRCT domains of MDC1 and BRCA1. Structure 18:167-176. PDF
Predicting the functional consequences of BRCA1 missense mutations.

Close to 100,000 women have now been screened for mutations in BRCA1 and a large database of mutations has been collected (for example, see the Breast Cancer Information Core Database, BIC:http://research.nhgri.nih.gov/bic/). This effort has uncovered hundreds of missense mutations throughout BRCA1 that have been very difficult to classify in terms of their cancer risks. We have undertaken structural and functional studies to classify the functional effects of all the know mutations within the BRCT region of BRCA1. Click here for a link to downloadable tables of our complete phospho-peptide binding, protein folding stability, and transcriptional activity data for 117 distinct BRCA1 BRCT missense mutations.

Figure 3. Predicting the functional consequences of BRCA1 missense mutations.
Coquelle, N., Green, R., Glover, J. N. M. (2011) Impact of BRCA1 BRCT Domain Missense Substitutions on Phosphopeptide Recognition. Biochemistry 50(21):4579-4589
Lee, M. S., Green, R., Marsillac, S. M., Coquelle, N., Williams, R. S., Yeung, T., Foo, D., Hau, D. D., Hui, B., Monteiro, A. N., Glover, J. N. M. (2010) Comprehensive analysis of missense variations in the BRCT domain of BRCA1 by structural and functional assays. Cancer Research 70(12):4880-4890.
Glover, J. N. M. (2006) Insights into the molecular basis of human hereditary breast cancer from studies of the BRCA1 BRCT domain. Familial Cancer, Special Issue Oncogenetics: Achievements and Challenges 5(1):89-93.
Williams, R. S., Chasman, D. I., Hau, D. D., Hui, B., Lau, A. Y. and Glover, J. N. M. (2003) Detection of protein folding defects caused by BRCA1-BRCT truncation and missense mutations. Journal Biological Chemistry 278(52):53007-53016. PDF
Williams, R. S. and Glover, J. N. M. (2003) Structural consequences of a cancer-causing BRCA1-BRCT missense mutation. The Journal of Biological Chemistry 278(4):2630-2635. PDF
The tandem BRCT repeat domain binds the C-terminus of the phosphorylated histone variant, γH2AX.

DNA double strand breaks are the most potentially dangerous form of genetic lesion. An early signal of a double strand break is the phosphorylation of H2AX within nucleosomes surrounding the break by DNA damage induced PI3 kinases such as ATM. These chromatin marks are specifically recognized by the BRCT protein MDC1. Our structural and functional work revealed that MDC1 not only recognizes the pSer residue, but also critically recognizes the γH2AX chain terminus.

Figure 4. The tandem BRCT repeat domain binds the C-terminus of the phosphorylated histone variant, γH2AX.
Campbell, S. J., Edwards, R. A. and Glover, J. N. M. (2010) Comparison of the structures and peptide binding specificities of the BRCT domains of MDC1 and BRCA1. Structure 18:167-176. PDF
Lee, M. S., Edwards, R. A., Thede, G. L. and Glover, J. N. M. (2005) Structure of the BRCT repeat domain of MDC1 and its specificity for the free COOH-terminal end of the gamma-H2AX histone tail. JBC 280(37):32053-32056. PDF
TopBP1 and coordination of the DNA replication stress response.

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Encounters between replicating polymerases and DNA damage can lead to an uncoupling of the polymerase and its DNA helicase as the polymerize stalls at the site of damage. This can lead to the exposure of large regions of single stranded DNA which in turn can result in aberrant DNA recombination. A key coordinator of a molecular response to replication stress is TopBP1, a multi-BRCT protein which coordinates protein-protein interactions at the fork to deal with this stress. We have studied the structures and functions of multiple BRCT domains with diverse targets and probed the functional consequences of these interactions with our collaborator, Junjie Chen. For example, we uncovered an unexpected interaction between TopBP1 and the DNA helicase and Fanconi anemia protein, BACH1/FancJ.

Figure 5.
Leung, C. C., Gong, Z., Chen, J., Glover, J. N. M. (2010) Molecular basis of BACH1/FANCJ recognition by TopBP1 in DNA replication checkpoint control. J Biol Chem. 2010 Dec 2. [Epub ahead of print]
Gong, Z., Kim, J.-E., Leung, C. C. Y., Glover, J. N. M. and Chen, J. (2010) BACH1/FANCJ acts with TopBP1 and participates early in DNA replication checkpoint control. Molecular Cell 37(3):438-446. PDF
Leung, C. C. Y., Kellogg, E., Kuhnert, A., Hänel, F., Baker, D. and Glover, J. N. M. (2010) Insights from the crystal structure of the sixth BRCT domain of Topoisomerase II-β binding protein 1. Protein Science 19(1):162-167. PDF
The BRCA1 partner, BARD1

BRCA1 forms a constitutive complex with BARD1, which, like BRCA1 also contains a C-terminal tandem BRCT domain. Our structure/function analysis of this protein revealed a divergent phospho-peptide binding cleft that may not interact with targets in the same manner as other BRCT proteins. BARD1, however, contacts the transcriptional machinery through an interaction involving a peptide region immediately N-terminal to the BRCT, and the transcriptional elongation factor, CstF-50.

Edwards, R. A., Lee, M. S., Tsutakawa, S. E., Williams, R. S., Nazeer, I., Kleiman, F.E., Tainer, J. A. and Glover, J. N. (2008) The BARD1 C-terminal domain structure and interactions with polyadenylation factor, CstF-50. Biochemistry 47(44):11446-11456. PDF