Quantitative analyses of RAG-RSS interactions and conformations revealed by atomic force microscopy

Jeffrey W. Pavlicek, Yuri L Lyubchenko, Yung Chang

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

During V(D)J recombination, site specific DNA excision is dictated by the binding of RAG1/2 proteins to the conserved recombination signal sequence (RSS) within the genome. The interaction between RAG1/2 and RSS is thought to involve a large DNA distortion that is permissive for DNA cleavage. In this study, using atomic force microscopy imaging (AFM), we analyzed individual RAG-RSS complexes, in which the bending angle of RAG-associated RSS substrates could be visualized and quantified. We provided the quantitative measurement on the conformations of specific RAG-12RSS complexes. Previous data indicating the necessity of RAG2 for recombination implies a structural role in the RAG-RSS complex. Surprisingly, however, no significant difference was observed in conformational bending with AFM between RAG1-12RSS and RAG1/2-12RSS. RAG1 was found sufficient to induce DNA bending, and the addition of RAG2 did not change the bending profile. In addition, a prenicked 12RSS bound by RAG1/2 proteins displayed a conformation similar to the one observed with the intact 12RSS, implying that no greater DNA bending occurs after the nicking step in the signal complex. Taken together, the quantitative AFM results on the components of the recombinase emphasize a tightly held complex with a bend angle value near 60°, which may be a prerequisite step for the site-specific nicking by the V(D)J recombinase.

Original languageEnglish (US)
Pages (from-to)11204-11211
Number of pages8
JournalBiochemistry
Volume47
Issue number43
DOIs
StatePublished - Oct 28 2008

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Atomic Force Microscopy
Protein Sorting Signals
Genetic Recombination
Conformations
Atomic force microscopy
DNA
Imaging techniques
VDJ Recombinases
Recombinases
V(D)J Recombination
DNA Cleavage
Proteins
Genes
Genome
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Quantitative analyses of RAG-RSS interactions and conformations revealed by atomic force microscopy. / Pavlicek, Jeffrey W.; Lyubchenko, Yuri L; Chang, Yung.

In: Biochemistry, Vol. 47, No. 43, 28.10.2008, p. 11204-11211.

Research output: Contribution to journalArticle

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