Identification and Characterization of Genes Involved in B-cell Specific Homologous Recombination

Jean-Marie Buerstedde
Department of Cellular Immunology
Heinrich-Pette-Institute
Martinistr. 52
D-20251 Hamburg, Germany
telephone: 0049 40 48051 290
fax: 0049 40 48051 296
email: buersted@genetics.hpi.uni-hamburg.de
prestype: Platform
presenter: Jean-Marie Buerstedde

Hiroshi Arakawa, Dima Lodygin, and Jean-Marie Buerstedde

Homologous recombination modifies the genome during meiotic and somatic cell development by enabling chomosomal crossovers and genetic exchanges between homologous sequences. It also plays a critical role for the repair of spontaneous and induced DNA damage. Gene conversion, as originally defined in yeast, is a special form of homologous recombination in which a donor sequence is duplicated and a target sequence is lost.

Homologous recombination plays a special role for the development of the immunoglobulin (Ig) gene repertoire, as B-cells in the chicken, rabbit and cow diversify their Ig genes at high frequency by gene conversion using pseudo V genes as donors. Ig gene conversion coexists with and resembles Ig somatic hypermutation in many vertebrate species.

The mechanism and enzymology of Ig gene conversion is poorly understood. To identify factors involved in this process we are establishing a comprehensive EST database from bursal B-cells which possess high Ig gene conversion activity. Blast searches of the already sequenced bursal ESTs against the public databases identified a number of promising candidates for DNA recombination factors. Among these are the genes encoding the DNA mismatch recognition factors MSH2, MSH3, MSH4 and MSH6, which have recently been implicated in Ig somatic hypermutation and switch recombination. In addition, we discovered a new type IV DNA polymerase (pol lambda) sharing homology to the patch repair DNA polymerase beta and the error prone terminal desoxytransferase. The pol lambda polymerase could either be involved in somatic hypermutation or in gene conversion.

Another interesting gene expressed in bursal B cells encodes a second structural homologue of the yeast RAD52 gene, which defines the double-strand break repair pathway by homologous recombination. We previously showed that the disruption of the first RAD52 gene in the chicken B cell line DT40 produces only a mild DNA repair defect, a finding which surprised many yeast geneticists. We now speculate that the second RAD52 homologue can compensate for the loss of the first RAD52 gene in vertebrate cells.

The exact functions of these genes are now determined by disruption in the DT40 cell line. DT40 is particularly suited, since it continues Ig gene conversion during cell culture and integrates transfected gene constructs at high ratios into the endogeneous loci. As we can recycle the our drug resistant marker by Cre-mediated excision, we shall also analyse the phenotype of multiple gene disruptions. We are for example planning to disrupt multiple MSH genes, DNA polymerases and RAD52 homologues within the same cell line to check for synergistic effects.

It is hoped that the isolation and characterization of B-cell specific recombination genes will eventually provide insight into the mechanism of immunoglobulin gene conversion. This research may also explain why targeted integration occurs at exceptionally high rates after transfection of DT40.



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