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Functional and genetic analysis of the BRCA1-PALB2 interaction

Srilatha Simhadri
M.S., Rutgers University - 2000

Thesis Advisor: Bing Xia, Ph.D.
Graduate Program in Cellular & Molecular Pharmacology

Rutgers Cancer Institute of New Jersey
Auditorium A
New Brunswick

Thursday, December 19, 2013
10:30 a.m.


PALB2, partner and localizer of BRCA2, interacts with the breast cancer tumor suppressors BRCA1 and BRCA2 to form a “BRCA” complex that promotes faithful DNA repair by homologous recombination and cell cycle checkpoint controls after DNA damage. PALB2 binds to the extreme N-terminus of BRCA2 via its C-terminal WD40 repeats, stabilizing BRCA2 and localizing it to chromatin and DNA damage-induced foci. PALB2 binds to BRCA1 through its N-terminal coiled-coiled domain, thereby mediating the physical and functional communication between BRCA1 and BRCA2.Through BRCA2, RAD51 is recruited to the same foci to engage in subsequent recombinational events. Mono-allelic mutations in PALB2 predispose toward development of familial breast, ovarian and pancreatic cancers. Bi-allelic mutations in PALB2 cause Fanconi anemia (FA), subtype N.
To define the physiological importance of BRCA1-PALB2 binding in vivo and to circumvent the embryonic lethality observed in mice resulting from a systemic knock-out of Palb2, we engineered, using a knock-in approach, a hypomorphic Palb2 allele (cc6) expressing a mutant PALB2 protein unable to bind BRCA1. Mice homozygous for the mutant allele are born in the expected Mendelian ratio. However, the cc6/cc6 males display impaired fertility, reduced testes size and gross testicular abnormalities. Histological examination revealed disruptions both in the architecture of the seminiferous tubules and in the progression of spermatogenesis. Analyses of meiotic chromosomes revealed a statistically significant defect in XY synapsis in the cc6/cc6 males, with no apparent defects either in autosomal synaptonemal complex formation or recombinational events. In addition, cells derived from mutant mice display mitomycin C-induced chromosomal instability, and the mice show increased spontaneous tumor development. Notably, the most affected organs are the uterine horns in cc6/cc6 females. Collectively, the mutant mice display features of Fanconi anemia.
PALB2, like BRCA1 and BRCA2, plays a key role at the G2/M checkpoint. Here, we show that PALB2-deficient FA cells exhibit a significant defect in G2/M checkpoint activation, which is corrected upon re-expression of wild-type protein. This function of PALB2 appears to be dependent on its binding to both BRCA1 and BRCA2, as PALB2 mutants abrogated for their BRCA1- or BRCA2-binding abilities are unable to correct the checkpoint activation failure. Furthermore, mouse embryonic fibroblasts and activated B cells derived from the Palb2-cc6 mice also exhibit a defect in G2/M checkpoint activation. Our results demonstrate that PALB2 plays an important role not only in the maintenance, but also in the activation of the G2/M checkpoint, and suggest that BRCA1, PALB2 and BRCA2 function together as a complex in the maintenance of genome integrity.

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