SLX4 as a Mediator of Crossover Pathway Decisions in Mammalian Meiosis
Principal Investigator: Paula Cohen
DESCRIPTION (provided by applicant):
The induction of hundreds of double strand breaks (DSB) during prophase I of meiosis initiates homologous
recombination (HR), which can result in the formation of crossovers (CO) that are essential for maintaining
chromosome interactions through until, and ensuring accurate segregation at, the first meiotic division. Only
10% of DSBs are destined to become COs, the others being repaired as non-crossovers (NCO), but all DSBs
must be repaired in a timely and robust fashion to prevent genome damage. Two distinct classes of COs can
occur: a major class I machinery, involving components of the DNA mismatch repair (MMR) pathway, and a
minor class II pathway, driven by the MUS81-EME1 endonuclease. The mechanisms by which selection of
these CO pathways, or NCO pathways, occurs remain unclear, although the placement and frequency of the
final CO tally must be stringently and exquisitely regulated to ensure accurate segregation at the first meiotic
division. In mouse, the Fanconi Anemia (FA) related protein, SLX4, is important for directing CO events
towards one of the two major pathways; mice lacking Slx4 exhibit a shift towards class I COs, loss of class II
COs, and persistent unrepaired DSBs at the end of prophase I, phenotypes similar to that of mice lacking
Mus81. This indicates that SLX4 may regulate class II CO events. SLX4 interacts with a large number of DNA
repair factors, including several structure specific endonucleases (SSEs; XPF-ERCC1, MUS81-EME1, SLX1),
as well as components of the FA and MMR pathways. In mouse, we have demonstrated that its interaction with
SLX1 is not critical for DSB repair, but that it interacts with the meiosis-specific MMR heterodimer, MutSγ.
Moreover, our studies indicate a functional interaction with BLM helicase, which regulates CO/NCO decisions
in late prophase I through the dissolution of double Holliday Junction (dHJ) repair intermediates. We have also
identified a novel interaction with another helicase in the FA pathway, FANCJ. The goal of the current
proposal is to elucidate the role SLX4 in driving different DSB repair pathways during prophase I, and
we hypothesize that this role depends on its interaction with key players in the repair network. In Aim 1,
we will explore the genetic interactions between BLM, SLX4, and MUS81, specifically asking whether SLX4 is
functioning to orchestrate class II events, or whether it is required to promote BLM-mediate dissolution of dHJs.
In Aim 2, we will identify key functional interactions involving SLX4 during prophase I, using elegant mouse
models to systematically interrogate each interacting partner of SLX4. In Aim 3, we will explore the roles and
co-dependence of FANCJ and SLX4 in meiotic recombination during prophase I, using our mutant mouse
models, combined with proteomics analysis, to understand how these two proteins interact functionally to
regulate CO/NCO decisions in the context of the different DNA repair pathways with which they interact.
Collectively, these studies represent the first functional analysis of the SLX4 interactome in mammalian
meiosis, and will illuminate how genome-wide CO is achieved through intersecting repair pathways.