A New NHS Plasmid System for Molecular Dissection of Mitotic
Recombination Pathways in Yeast.
Carlo V. Bruschi, Valentina Caputo
Microbiology Group, ICGEB, AREA Science Park, Trieste, I-34012, Italy
Ensuing double-strand break (DSB) repair in eukaryotes, crossover (CO)
or non-crossover (gene conversion, GC), non-homologous end-joining
(NHEJ) and single-strand annealing (SSA) events, up to now have been
studied separately, although it is clear that all of them can occur
simultaneously in different proportions. In order to understand the
overall dynamics of DSB repair, we have created a new molecular system
to simultaneously detect all four types of recombinational DBS repair.
To this end, we constructed an in vivo/in vitro HNS
plasmid system (HNS: HR, NHEJ, SSA), based upon two topologically
different DNA molecules, which allows us to follow all four
recombination processes at once. The plasmids, named pURRA8 and pRURA8,
contain two truncated non-functional URA3 genes in direct or inverted
orientation respectively, sharing a central homologous region where a I-SceI DBS is introduced artificially. The plasmids also carry a
centromere sequence and two phenotypic markers TRP1 and
ADE8. The HNS system has been used to identify new genes specific
for the various recombination pathways. DBS were induced in vivo
by the expression of the SceI endonuclease under Gal1
promoter control. After transformation, we performed transposon
mutagenesis using the mTn-lacZ/LEU2 library system and selected cells
that lost the ability to recombine. With the HNS system, several genes
have been identified by transposon mutagenesis and are currently under
study.
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