Abstract
Lymphoid cells of the vertebrate immune system rely on factors in the non-homologous end-joining (NHEJ) DNA repair pathway to form signal joints during V(D)J recombination. Unlike other end-joining reactions, signal joint formation is a specialized case of NHEJ that also requires the lymphoid-specific RAG proteins. Whether V(D)J recombination requires the Mre11-Rad50-Nbs1 complex remains an open question, as null mutations in any member of the complex are lethal in mammals. However, Saccharomyces cerevisiae strains carrying null mutations in components of the homologous Mre11p-Rad50p-Xrs2p (MRX) complex are viable. We therefore took advantage of a recently developed V(D)J recombination assay in yeast to assess the role of MRX in V(D)J joining. Here we confirmed that signal joint formation in yeast is dependent on the same NHEJ factors known to be required in mammalian cells. In addition, we showed an absolute requirement for the MRX complex in signal joining, suggesting that the Mre11-Rad50-Nbs1 complex may be required for signal joint formation in mammalian cells as well.
Highlights
The immune systems of jawed vertebrates assemble a diverse array of immunoglobulin molecules and T-cell receptors by the process known as V(D)J recombination (1)
We extended and confirmed these results through an analysis of the genetic requirements for signal joint formation and RAG-mediated transposition in a series of isogenic yeast strains with disruptions in genes involved in non-homologous end joining (NHEJ) (YKU70, LIG4, MRE11, RAD50, XRS2, and NEJ1), homologous recombination (RAD52) (58), or interstrand cross-link repair (PSO2)
That work determined that the yeast XRCC4 homologue, LIF1, is necessary for precise signal joint formation in yeast but is dispensable for RAGmediated strand transfer
Summary
Strains with disruptions in DNA repair genes were made by standard PCR-based gene replacement techniques. Strains MAV109, MAV134, MAV135-2, MAV144, MAV288, MAV312, MAV320, and MAV321 were derived from parental strain BY4704 by replacing the NEJ1, LIG4, YKU70, PSO2, RAD50, RAD52, MRE11, or XRS2 open reading frames, respectively, with the kanMX4 cassette. The resulting strains were each transformed with the RAG expression plasmids
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