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Biochemistry
of homologous recombination 
Characterization
of recombinase function in vivo
Characterization
of DNA intermediates in meiotic recombination
Genetic dissection
of BRCA1 function
Intergenic suppression
of BRCA1 function
Characterization
of drugs that alter the efficiency of homologous recombination
Although detection of DNA
intermediates in vivo has revealed a number of mechanistic features
of meiotic recombination, several questions about the mechanism
remain. Until recently it was thought that the two major types
of meiotic recombinants, crossovers and non-crossovers, arise
via a common intermediate called the double Holliday junction.
However, analysis of the timing and genetic requirements for formation
of crossover and non-crossover products relative to the timing
of appearance and disappearance of double Holliday junctions suggested
that non-crossovers can arise without forming double Holliday
junction intermediates. Additional studies from fission yeast
indicate that recombinants can also arise via single rather than
double Holliday junctions. These findings seem to indicate that
the array recombination products in a single meiotic cell results
from a collection of related recombination mechanisms rather than
from a single mechanism. We hope to better understand this complexity
by developing methods capable of detecting recombination intermediates
that are diagnostic of a particular recombination mechanism. For
example, non-crossover recombination intermediates have been proposed
to form via the synthesis-dependent strand-annealing pathway (SDSA).
We have developed a genetic method, called “ends-apart,”
that is designed to detect the genetic signature of SDSA. The
results obtained with ends-apart provide strong evidence that
SDSA does contribute to non-crossover recombination. Ends-apart
is currently being used to help identify genes whose function
is specific to SDSA.
Various different
pathways for homologous recombination that have been proposed
to occur during meiosis in budding yeast. Lines represent
DNA single strands, green represents regions of newly synthesized
DNA. (
Bishop DK. Multiple mechanisms of meiotic recombination. Cell.
2006 Dec 15;127(6):1095-7.)
Another approach to studying
the mechanism of recombination involves use of the two-dimensional
gel system developed by Schwacha and Kleckner to study Dmc1’s
role recombination partner choice. Initiation of recombination
by formation of a DNA double strand break can lead to engagement
of either a sister chromatid or a homologous chromatid. In mitosis
the sister is almost always chosen to be the partner while in
meiosis, the homologue must be chosen in order for the recombination
event to fulfill a critical function of meiotic recombination:
forming the connections between chromosomes required for their
accurate reductional segregation. We are using the 2-D gel method
to determine the role of Dmc1 accessory factors in homologue partner
choice.
Southern blot of
a 2-D gel designed to detect recombination intermediates at a
recombination Hotspot. IH=Inter-homologue double Holliday
junction. IS= IH=Inter-sister double Holliday junction SEI=single
end intermediate. (Method described in Hunter and Kleckner, Cell
2001 Jul 13;106(1):59-70).
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