Identification of chromatin-related protein interactions using protein microarrays.

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.

RAD51C interacts with RAD51B and is central to a larger protein complex in vivo exclusive of RAD51.

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.