DISSECTING THE ROLE OF BRCA2, RAD51 AND SMARCAL1 IN VERTEBRATE CHROMOSOMAL DNA REPLICATION.

DNA replication is a fundamental macromolecular event that is essential for cell division. During each cell cycle the entire genome has to be precisely duplicated to ensure genome integrity and stability. In the process of DNA replication, replication forks encounter endogenous and exogenous lesions and these lesions have to be rectified to transfer stable genetic material to daughter cells. Emerging evidences connected a role for Homologous Recombination (HR) proteins to replication fork protection during unperturbed DNA replication. Since HR protein, BRCA2 and Rad51 are essential for cell survival we used Xenopus laevis cell-free extract to dissect the function of BRCA2 and Rad51 during chromosomal DNA replication. Using Electron Microscopy (EM) we show that BRCA2 and Rad51 function together to prevent single-stranded DNA (ssDNA) accumulation at and behind the forks during unperturbed DNA replication. We further discovered that BRCA2 mediates interaction between Rad51, Polymerase alpha (α) and Polymerase delta (δ) and this interaction likely promote efficient re-priming and polymerising activity at stalled replication forks to prevent ssDNA accumulation. Moreover we show that inhibition of replicative polymerases in the absence of Rad51 results in increased frequency of replication fork reversal activity. We further found that replication fork reversal is predominantly induced by an annealing helicase SMARCAL1 in the absence of Rad51. Collectively our findings indicate that, to prevent ssDNA accumulation and aberrant replication fork architecture, timely re-priming of Polymerase alpha mediated by Rad51 is essential. Hence, loss of Rad51 impact replication fork architecture, eventually resulting in chromosomal abnormalities.