RAD51B and RAD51C are two of five known paralogs of the human RAD51 protein that are thought to function in both homologous recombination and DNA double-strand break repair. This work describes the in vitro and in vivo identification of the RAD51B/RAD51C heterocomplex. The RAD51B/RAD51C heterocomplex was isolated and purified by immunoaffinity chromatography from insect cells co-expressing the recombinant proteins. Moreover, co-immunoprecipitation of the RAD51B and RAD51C proteins from HeLa, MCF10A, and MCF7 cells strongly suggests the existence of an endogenous RAD51B/RAD51C heterocomplex. We extended these observations to examine the interaction between the RAD51B/RAD51C complex and the other RAD51 paralogs. Immunoprecipitation using protein-specific antibodies showed that RAD51C is central to a single large protein complex and/or several smaller complexes with RAD51B, RAD51D, XRCC2, and XRCC3. However, our experiments showed no evidence for the inclusion of RAD51 within these complexes. Further analysis is required to elucidate the function of the RAD51B/RAD51C heterocomplex and its association with the other RAD51 paralogs in the processes of homologous recombination and DNA double-strand break repair.The human RAD51 protein functions in homologous recombination and DNA double-strand break repair (1-4). Five paralogs of human RAD51 have been identified: RAD51B (hREC2, RAD51L1), RAD51C (RAD51L2), RAD51D (RAD51L3), XRCC2, and XRCC3; these proteins share ϳ25% amino acid sequence identity with one another and RAD51 (5-9). DT40 chicken cell knockouts have been generated for each paralog, all of which exhibit a lack of RAD51 foci formation as well as enhanced radiation and cisplatin sensitivity, consistent with a deficiency in recombinational repair (10, 11). These results are consistent with previous work, which showed that the XRCC3 knockout CHO cell line (irs1SF) is deficient in RAD51 foci formation, suggesting that XRCC3 is required for RAD51 function (12). Similarly, XRCC2-defective cell lines also fail to form damage-dependent RAD51 foci (13). Furthermore, both XRCC2 and XRCC3 have been shown to be required for repair of double-strand breaks by homologous recombination in vivo (14 -16). Initially by sequence and now by association and function, it has become increasingly evident that these RAD51 paralogs participate together in recombination and repair processes.Extensive yeast two-and three-hybrid analysis suggests that there are a variety of putative protein-protein interactions between the RAD51 paralogs. These include XRCC2/RAD51D (17, 18) and XRCC3/RAD51(19). Moreover, interactions have been suggested between RAD51C/RAD51B, RAD51C/RAD51D, RAD51C/XRCC3, and RAD51C/RAD51 (8, 18). More recent biochemical evidence has corroborated the interaction between XRCC2 and RAD51D by co-purification of the recombinant proteins and co-elution of the native proteins by gel filtration from mammalian cell extracts; RAD51D was further shown to be a DNA-stimulated ATPase (17). In addition, a stable heterocomplex was demons...

When the beta(5) (short form) and gamma(2) subunits of heterotrimeric G proteins were expressed with hexahistidine-tagged alpha(i) in insect cells, a heterotrimeric complex was formed that bound to a Ni-NTA-agarose affinity matrix. Binding to the Ni-NTA-agarose column was dependent on expression of hexahistidine-tagged alpha(i) and resulted in purification of beta(5)gamma(2) to near homogeneity. Subsequent anion-exchange chromatography of beta(5)gamma(2) resulted in resolution of beta(5) from gamma(2) and further purification of beta(5). The purified beta(5) eluted as a monomer from a size-exclusion column and was resistant to trypsin digestion suggesting that it was stably folded in the absence of gamma. beta(5) monomer could be assembled with partially purified hexahistidine-tagged gamma(2) in vitro to form a functional dimer that could selectively activate PLC beta2 but not PLC beta3. alpha(o)-GDP inhibited activation of PLC beta2 by beta(5)gamma(2) supporting the idea that beta(5)gamma(2) can bind to alpha(o). beta(5) monomer and beta(5)gamma(2) only supported a small degree of ADP ribosylation of alpha(i) by pertussis toxin (PTX), but beta(5) monomer was able to compete for beta(1)gamma(2) binding to alpha(i) and alpha(o) to inhibit PTX-catalyzed ADP ribosylation. These data indicate that beta(5) functionally interacts with PTX-sensitive GDP alpha subunits and that beta(5) subunits can be assembled with gamma subunits in vitro to reconstitute activity and also support the idea that there are determinants on beta subunits that are selective for even very closely related effectors.

In previous work (Sankaran, B., Osterhout, J., Wu, D., and Smrcka, A. V. (1998) J. Biol. Chem. 273, 7148 -7154), we showed that overlapping peptides, N20K (Asn 564 -Lys 583 ) and E20K (Glu 574 -Lys 593 ), from the catalytic domain of phospholipase C (PLC) ␤2 block G␤␥-dependent activation of PLC ␤2. The peptides could also be directly cross-linked to ␤␥ subunits with a heterobifunctional cross-linker succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate. Cross-linking of peptides to G␤ 1 was inhibited by PLC ␤2 but not by ␣ i1 (GDP), indicating that the peptide-binding site on ␤ 1 represents a binding site for PLC ␤2 that does not overlap with the ␣ i1 -binding site. Here we identify the site of peptide cross-linking and thereby define a site for PLC ␤2 interaction with ␤ subunits. Each of the 14 cysteine residues in ␤ 1 were altered to alanine. The ability of the PLC ␤2-derived peptide to cross-link to each ␤␥ mutant was then analyzed to identify the reactive sulfhydryl moiety on the ␤ subunit required for the cross-linking reaction. We find that C25A was the only mutation that significantly affected peptide cross-linking. This indicates that the peptide is specifically binding to a region near cysteine 25 of ␤ 1 which is located in the amino-terminal coiled-coil region of ␤ 1 and identifies a PLC-binding site distinct from the ␣ subunit interaction site.Guanine nucleotide-binding proteins (G proteins) 1 are a large group of structurally similar proteins consisting of three subunits (␣, ␤, and ␥) that are central molecules coupling seven-transmembrane domain-spanning receptors to downstream effector molecules. Activation of G proteins begins with a ligand-induced conformational change of the receptor which catalyzes the release of GDP from the ␣ subunit in exchange for GTP (1, 2). In the GDP-bound heterotrimeric state, ␣(GDP)⅐␤␥, neither ␣(GDP) nor ␤␥ can regulate effector activity. Upon receptor-catalyzed G protein activation, the heterotrimer dissociates into free ␣(GTP) and free ␤␥ subunits. It is well understood that both ␣(GTP) and ␤␥ subunits can interact with a variety of downstream effector molecules including enzymes and ion channels. GTP is hydrolyzed to GDP, and reassociation of ␣(GDP) with ␤␥ results in deactivation of ␤␥-dependent signaling. Despite detailed knowledge of ␣-and ␤␥ subunit functions, the mechanism for how ␤␥ subunits activate its variety of effectors is not entirely understood.Effector-binding sites on the surface of ␤␥ are beginning to be mapped. The putative competition between ␣(GDP) and effectors for ␤␥ forms the premise for recent studies to map effectorbinding sites at the ␣ subunit-binding interface on ␤. The three-dimensional structure of the G protein heterotrimer reveals that the ␤ subunit is a ␤-propeller with seven "blades" and an amino-terminal ␣-helix (3, 4). The ␣ subunit binds to a portion of the top of the ␤-propeller and along side one of the blades of the propeller. Two groups have shown that alanine substitution of ␣-contacting residues on the top surface...

Dynamic structural changes in chromatin are mediated by protein interactions that modulate multiple cellular processes including replication, transcription, recombination and DNA repair. Complexes that recognize chromatin are defined by several distinct groups of proteins that either directly modify histones or interact with histone-DNA complexes. A protein microarray format was used to analyze the interaction of various DNA repair proteins with chromatin components. We applied proteins, antibodies and DNA to functionalized glass slides and interrogated the slides with our proteins of interest to identify novel protein-protein interactions for proteins involved in DNA double-strand break repair. Here we demonstrate that the DNA repair protein RAD51B, and not its cognate partner RAD51C, interacts with histones and not nucleosomes. Nucleosome-specific interactions were demonstrated with the recently identified SWI/SNF protein, SMARCAL1. Unique RAD51B-histone interactions were corroborated using Far Western analysis. This is the first demonstration of an interaction between RAD51B and histone proteins that may be important for the successful repair of DNA double-strand breaks.