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. 2006 Apr 28;281(17):11649-57.
doi: 10.1074/jbc.M601249200. Epub 2006 Mar 2.

Recombination mediator and Rad51 targeting activities of a human BRCA2 polypeptide

Joseph San Filippo  1 , Peter Chi, Michael G Sehorn, Julia Etchin, Lumir Krejci, Patrick Sung
Affiliations

Recombination mediator and Rad51 targeting activities of a human BRCA2 polypeptide

Joseph San Filippo et al. J Biol Chem. .

Abstract

BRCA2 likely exerts its tumor suppressor function by enhancing the efficiency of the homology-directed repair of injured chromosomes. To help define the DNA repair role of BRCA2, we expressed and purified a polypeptide, BRC3/4-DBD, that harbors its BRC3 and BRC4 repeats and DNA binding domain. BRC3/4-DBD interacted with hRad51 and bound DNA with a distinct preference for single-stranded (ss) DNA. Importantly we demonstrated by biochemical means and electron microscopy that BRC3/4-DBD nucleates hRad51 onto ssDNA and acts as a recombination mediator in enabling hRad51 to utilize replication protein A-coated ssDNA as recombination substrate. These functions of BRC3/4-DBD required both the BRC repeats and the BRCA2 DNA binding domain. The results thus clarify the role of BRCA2 in Rad51-dependent DNA recombination and repair, and the experimental strategies described herein should be valuable for systematically deciphering this BRCA2 function.

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Figures

FIGURE 1
FIGURE 1. Purification of BRC3/4-DBD
A, schematic outlining the functional domains in BRCA2. The DBD consists of a helical domain (HD) and three oligonucleotide binding (OB1, OB2, and OB3) folds that confer the ability to interact with DSS1 and ssDNA. In addition to the BRC repeats, the carboxyl terminus possesses a distinct Rad51 binding domain. B, the human BRCA2-derived polypeptide BRC3/4-DBD harbors two of the BRC repeats (BRC3 and BRC4 within residues 1409–1596) linked to the DBD (residues 2477–3194). BRC3/4-DBD contains thioredoxin (Trx) and S tags at its amino terminus and a His6 tag at its carboxyl terminus. C, schematic of the chromatographic procedure devised for BRC3/4-DBD purification. D, purified BRC3/4-DBD was analyzed by SDS-PAGE and Coomassie Blue staining (2 μg in lane 2) or immunoblotting with anti-histidine antibodies (200 ng in lane 3). E, results from MALDI-TOF analysis of purified BRC3/4-DBD. Six representative fragments and their corresponding e values are shown. aa, amino acids.
FIGURE 2
FIGURE 2. Analysis of proteins by SDS-PAGE and Coomassie Blue staining
Lane 2, hRad51; lane 3, hRad51 K133R; lane 4, RecA; lane 5, GST-BRC3/4; lane 6, BRCA2-DBD; lane 7, hRPA (RPA70, RPA34, and RPA14 subunits); lane 8, SSB.
FIGURE 3
FIGURE 3. BRC3/4-DBD binds hRad51
A, BSA, hRad51, hRad51 K133R, and RecA were mixed with anti-S protein-agarose beads in the absence (lanes 1–12) or presence of BRC3/4-DBD (lanes 13–24) and then subjected to affinity pull-down with anti-S-agarose beads. B, GST-BRC3/4, DBD, and BRC3/4-DBD were subjected to affinity pull-down with Affi beads conjugated to BSA (Affi-BSA; lanes 1–9) or hRad51 (Affi-hRad51; lanes 10–21). C, pull-down assays using GST or GST-BRC3/4 on glutathione-Sepharose confirmed that BRC3/4 binds Rad51 but not RecA. The supernatant (S), wash (W), and SDS eluate (E) from the above reactions were analyzed by SDS-PAGE and Coomassie Blue staining.A proteolytic product of BRC3/4-DBD is marked by the asterisk.
FIGURE 4
FIGURE 4. BRC3/4-DBD has high affinity for ssDNA
A and B, increasing amounts of purified BRC3/4-DBD (20–500 nm, lanes 2–9) were incubated with 30 nm​ 32P-labeled ssDNA or dsDNA and then analyzed. Treatment of the nucleoprotein with SDS and proteinase K (SDS/PK) released the DNA substrate (lane 10). C, BRC3/4-DBD (50–500 nm) was incubated with the mixture of 32P-labeled ssDNA and dsDNA and then analyzed. D, the results from the experiment in C are plotted.
FIGURE 5
FIGURE 5. BRC3/4-DBD targets hRad51 to ssDNA
A, schematic of the assay. Magnetic bead-bound oligo(dT) was incubated with hRad51, BSA, and BRCA2-derived polypeptides, without or with an excess of dsDNA, as indicated. Proteins bound to the oligo(dT) were captured with a magnet and then eluted with SDS. B, the supernatants (super) and SDS eluates (beads) were analyzed for their protein and DNA contents. Although the majority of hRad51 was trapped on the dsDNA as expected (lane 3), BRC3/4-DBD efficiently targeted hRad51 to the ssDNA (lanes 4 and 5). GST-BRC3/4 (BRC3/4) or DBD was ineffective in this regard (lanes 6 and 7, respectively). C, the bead-bound fractions (from lanes 1, 3, and 5 in B) were examined for hRad51-mediated ATP hydrolysis. D, Rad51-mediated ATP hydrolysis was assayed in the presence of ssDNA and with or without BRC3/4-DBD.
FIGURE 6
FIGURE 6. Recombination mediator activity of BRC3/4-DBD
A and B, homologous DNA pairing reactions containing hRad51 or hRad51 K133R and varying amounts of BRC3/4-DBD were carried out with ssDNA or hRPA-coated ssDNA as indicated. The averaged values of results from three independent experiments are presented in the histograms. The no protein control (lane 1) is marked as “blank,” and ATP was omitted from the reaction in lane 10.
FIGURE 7
FIGURE 7. Effects of GST-BRC3/4 (A) and DBD (B) on homologous DNA pairing by hRad51 K133R
The reactions were carried out exactly as described for Fig. 6 but without hRPA.
FIGURE 8
FIGURE 8. Experiments showing that neither GST-BRC3/4 (A), DBD (B), nor a mixture of the two polypeptides (C) is capable of overcoming the inhibitory effect of hRPA on homologous DNA pairing by hRad51 K133R
The concentrations of the BRCA2 polypeptides were 25, 50, 100, 150, and 200 nm.
FIGURE 9
FIGURE 9. Examination of recombination mediator activity by electron microscopy
Examples of hRad51 K133R-ssDNA nucleoprotein filaments (A) and hRPA-ssDNA complexes (B) are shown (arrows). Control experiments confirmed the requirement for ssDNA in the formation of the hRad51 K133R filaments and hRPA-containing complexes. The bar in black denotes a length of 50 nm. C, data quantification of reaction mixtures that contained either hRad51 K133R and hRPA or hRad51 K133R, hRPA, and BRC3/4-DBD. Over 1,500 nucleoprotein complexes were counted to determine the relative abundance of the hRad51 K133R-ssDNA filaments.
FIGURE 10
FIGURE 10. Specificity and versatility of the BRC3/4-DBD recombination mediator activity
A, BRC3/4-DBD is unable to overcome the suppressive effect of hRPA on RecA-mediated homologous DNA pairing (lanes 6–9). hRad51 K133R (hRad51KR) was included in this analysis as control (lanes 2–5). B, E. coli SSB attenuates homologous DNA pairing by hRad51 K133R, and this suppressive effect is overcome by BRC3/4-DBD (lanes 4–9). The no protein control (lane 1) is marked as “blank.” The results are presented in the histograms.
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References

    1. Jasin M. Oncogene. 2002;21:8981–8993. - PubMed
    1. D'Andrea AD. Genes Dev. 2003;17:1933–1936. - PubMed
    1. Symington LS. Microbiol. Mol. Biol. Rev. 2002;66:630–670. - PMC - PubMed
    1. Sung P, Krejci L, Van Komen S, Sehorn MG. J. Biol. Chem. 2003;278:42729–42732. - PubMed
    1. Wooster R, Bignell G, Lancaster J, Swift S, Seal S, Mangion J, Collins N, Gregory S, Gumbs C, Micklem G. Nature. 1995;378:789–792. - PubMed

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