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Nucleic Acids Res. 2003 Apr 1;31(7):2014-23.

Self-association and conformational properties of RAG1: implications for formation of the V(D)J recombinase.

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1
Department of Biochemistry and Molecular Biology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA.

Abstract

RAG1 and RAG2 catalyze the initial DNA cleavage steps in V(D)J recombination. Fundamental properties of these proteins remain largely unknown. Here, self-association and conformational properties of murine core RAG1 (residues 384-1008) were examined. As determined by multi-angle laser light scattering measurements, the molecular masses of two predominant core RAG1 species corresponded to dimeric and tetrameric states. Similar results were obtained using a RAG1 fragment containing residues 265-1008, indicating that a non-core portion of RAG1 does not alter the oligomerization states observed for the core region. The fraction of core RAG1 in the tetrameric state increased significantly at lower ionic strengths (0.2 versus 0.5 M NaCl), indicating that this oligomeric form may factor into the physiological function of RAG1. In addition, the secondary structural content of core RAG1, obtained by circular dichroism spectroscopy, demonstrated a significant dependence on ionic strength with a 26% increase in alpha-helical content from 0.2 to 1.0 M NaCl. Together, these results indicate that structural and oligomerization properties of core RAG1 are strongly dependent on electrostatic interactions. Furthermore, the secondary structure of core RAG1 changes upon binding to DNA, with larger increases in alpha-helical content upon binding to the recombination signal sequence (RSS) as compared with non-sequence-specific DNA. As shown by electrophoretic mobility shift assays, higher order oligomeric forms of core RAG1 bound to the canonical RSS. Furthermore, core RAG2 (residues 1-387) formed complexes with multimeric RAG1 species bound to a single RSS, providing additional support for the physiological relevance of higher order oligomeric states of RAG1.

PMID:
12655019
PMCID:
PMC152797
[Indexed for MEDLINE]
Free PMC Article
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