During lymphoid development, functional immunoglobulin and T cell receptor genes are assembled from gene segments by a process called V(D)J recombination, which is essential for generating the diversity of the immune response. In the first stage of this reaction, specific double-strand breaks are made at the target sites (RSS) by the RAG1/RAG2 protein complex. The later steps necessary for rejoining employ many protein factors that are also used for repair of X-ray damage. We continue to investigate both halves of the pathway. 1) After coupled cleavage of complementary RSSs, virtually all of the cleaved RSS ends remained associated with RAG1/2 in a stable signal end complex. This complex was distinct from various pre-cleavage RAG1/2-RSS complexes in that only after cleavage was it resistant to treatment with heparin. A mammalian joining apparatus consisting of purified Ku70/86, XRCC4 and DNA ligase IV, all required for joining of cleaved ends in vivo, was sufficient to join deproteinized cleaved ends. Retention of RSSs within this complex blocked access to the cleaved DNA ends so that they could not be joined together by the mammalian joining apparatus or by T4 ligase. Sequestration of cleaved RSS ends within this complex would account for the persistence of the broken ends in the cell after cleavage and may explain why they do not normally activate the DNA-damage dependent cell cycle checkpoint. 2) The tumor suppressor BRCA1 plays an important role in protecting mammalian cells against genomic instability, but little is known about its modes of action. In this work we demonstrate that recombinant human BRCA1 protein binds strongly to DNA, an activity conferred by a domain in the center of the Brca1 polypeptide. As a result of this binding, BRCA1 inhibits the nucleolytic activities of the Mre11/Rad50/Nbs1 complex, an enzyme implicated in numerous aspects of double-strand break repair. BRCA1 displays a preference for branched DNA structures and forms protein-DNA complexes cooperatively between multiple DNA strands, but without DNA sequence specificity. This fundamental property of BRCA1 may be an important part of its role in DNA repair and transcription.
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