YEARS

-

AUTHORS

Martin F. Gellert

TITLE

Structural studies of the post-cleavage complex in V(D)J recombination

ABSTRACT

Although there is a great deal of biochemical information about the RAG1and RAG2 proteins, no correlated structural data exist so far. As a result of previous work from this group, we identified the best-defined and most stable complex in this system as being the complex RAG1 and RAG2 make with two DNA ends after cutting DNA at the recombination sites. This complex has now been prepared in sufficient quantity for high-resolution electron microscopy. A collaborative arrangement with the groups of Dr. Wei Yang (LMB) and Dr. Alasdair Steven (NIAMS) has produced for the first time pictures of negatively stained samples good enough for image reconstruction, which shows evidence of a hollow core and other defined features. Present efforts focus on higher resolution that can be obtained with cryo-electron microscopy and immuno-electron microscopy, to be combined with molecular weight estimation by scanning transmission electron microscopy. Together with biophysical measurements, the results have already clarified the issue of the protein stoichiometry of the complex, which has been in dispute in the literature. Further scaling up of the protein preparation method may also permit crystallization of the complex.

FUNDED PUBLICATIONS

  • Requirements for DNA hairpin formation by RAG1/2.
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    16 TRIPLES      15 PREDICATES      16 URIs      7 LITERALS

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    1 grants:8d3f87367b03c6a4bc1b679aeb39f9e3 sg:abstract Although there is a great deal of biochemical information about the RAG1and RAG2 proteins, no correlated structural data exist so far. As a result of previous work from this group, we identified the best-defined and most stable complex in this system as being the complex RAG1 and RAG2 make with two DNA ends after cutting DNA at the recombination sites. This complex has now been prepared in sufficient quantity for high-resolution electron microscopy. A collaborative arrangement with the groups of Dr. Wei Yang (LMB) and Dr. Alasdair Steven (NIAMS) has produced for the first time pictures of negatively stained samples good enough for image reconstruction, which shows evidence of a hollow core and other defined features. Present efforts focus on higher resolution that can be obtained with cryo-electron microscopy and immuno-electron microscopy, to be combined with molecular weight estimation by scanning transmission electron microscopy. Together with biophysical measurements, the results have already clarified the issue of the protein stoichiometry of the complex, which has been in dispute in the literature. Further scaling up of the protein preparation method may also permit crystallization of the complex.
    2 sg:fundingAmount 1087348.0
    3 sg:fundingCurrency USD
    4 sg:hasContribution contributions:9c222655b8cab6b88a25d6c02675199c
    5 sg:hasFieldOfResearchCode anzsrc-for:02
    6 anzsrc-for:0299
    7 sg:hasFundedPublication articles:dbe1c28c30de48b51b951db7c7e8ec9f
    8 sg:hasFundingOrganization grid-institutes:grid.419635.c
    9 sg:hasRecipientOrganization grid-institutes:grid.419635.c
    10 sg:language English
    11 sg:license http://scigraph.springernature.com/explorer/license/
    12 sg:scigraphId 8d3f87367b03c6a4bc1b679aeb39f9e3
    13 sg:title Structural studies of the post-cleavage complex in V(D)J recombination
    14 sg:webpage http://projectreporter.nih.gov/project_info_description.cfm?aid=7734112
    15 rdf:type sg:Grant
    16 rdfs:label Grant: Structural studies of the post-cleavage complex in V(D)J recombination
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