Survival pathways supporting BRCA1 function.

Background: The genes involved in the most accurate form of DNA repair, homologous recombination, are needed for normal development and normal cellular recombination. In recent years it has become clear that the proteins have multiple functions that contribute to the outcomes of good DNA repair under different stress contexts.
We are particularly interested in one of the most significant DNA repair genes, BRCA1, which was first discovered due to its association with breast cancer. In recent years we have started to prise apart the subtly different functions encoded in this one gene and it is now clear that it performs at least three different functions. These functions are likely to contribute to its full role in promoting normal development and normal cellular processes, and one in particular is needed as cells are replicating, protecting the exposed DNA ends when replication stalls.
Objectives: In this project we will use cells in which we have disabled the replication fork protection function of the BRCA1. We aim to use genome-wide CRISPR-Cas9 based gene knock-out to examine what cellular pathways support cell survival in the absence of that one function.
Methods: The CRISPR screens include a bar-code based sequencing of cells to establish the identity of knocked-out genes, combined with pathway analysis to establish which cellular signalling pathways have been lost. The findings will be validated by specifically removing key nodes of the newly identified pathways using RNA silencing to establish that cells lacking the specific BRCA1 function specifically require the identified pathway nodes.
By these means we hope to understand how BRCA1 enables normal development and promotes critical cellular functions such as DNA replication.