Abstract
BackgroundThe breast cancer susceptibility protein, BRCA1 functions to maintain the integrity of the genome. The exact mechanisms by which it does so, however, remain unclear. The ability of BRCA1 to bind directly to DNA suggests a more direct role. However, little research has been conducted to understand the functional relevance of this characteristic of BRCA1. In this study we examine the DNA substrate specificity of BRCA1 and how this may be controlled by one of its interacting partners, p53.Methodology/Principal FindingsUsing competition gel retardation assays we have examined the ability of residues 230-534 of BRCA1 to discriminate between different synthetic DNA substrates that mimic those recognised by the DNA damage response i.e. four-way junction DNA, mismatch containing DNA, bulge containing DNA and linear DNA. Of those tested the highest affinity observed was for four-way junction DNA, with a 20 fold excess of each of the other synthetic DNA's unable to compete for any of the bound BRCA1 230-534. We also observed a higher affinity for C∶C and bulge containing DNA compared to linear duplex and G∶T containing DNA. BRCA1 230-534 also has interaction sites for the tumour suppressor p53 and we show that titration of this complex into the DNA binding assays significantly reduces the affinity of BRCA1 for DNA.Conclusions/SignificanceIn this paper we show that BRCA1 can discriminate between different types of DNA damage and we discuss the implications of this with respect to its function in DNA repair. We also show that the DNA binding activity can be inhibited by the tumour suppressor p53 and suggest that this may prevent genome destabilizing events such as HR between non-homologous sequences.
Highlights
Cellular and environmental agents that interact with and modify the chemical structure of DNA constantly undermine the integrity of the human genome
The BRCA1 Associated Genome Surveillance Complex (BASC) complex is characterised by a high number of factors that are involved in the DNA damage response, for example the mismatch repair complexes MLH1-PMS2 and MSH2–MSH6, the NBS1MRE11-RAD50 complex which participates in pathways associated with the repair of double strand breaks and the global damage sensor ATM
In particular we have synthesised substrates that mimic those formed during the repair of double strand breaks by homologous recombination and those recognised by the mismatch repair machinery (G:T, C:C and bulge containing DNA), as BRCA1 is known to participate in these pathways [2,25]
Summary
Cellular and environmental agents that interact with and modify the chemical structure of DNA constantly undermine the integrity of the human genome. The BRCA1 protein, which is associated with hereditary breast and ovarian cancer, acts as a scaffold that both organises and coordinates a number proteins involved in maintaining genome integrity [1,2]. Largely unfolded [12,13] the central region of BRCA1 has interaction sites for a number of proteins that are associated with maintaining genome integrity (e.g. p53 [14], RAD50 [15], FANCA [16], BRCA2 [17]) and distorted DNA structures that occur as a result of the repair of double strand breaks by homologous recombination [18,12]. In this study we examine the DNA substrate specificity of BRCA1 and how this may be controlled by one of its interacting partners, p53
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