Department of Cellular Immunology
D-20251 Hamburg, Germany
telephone: 0049 40 48051 290
fax: 0049 40 48051 296
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
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
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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