http://worldcat.org/entity/work/id/553334420

A genome-wide siRNA screen reveals diverse cellular processes and pathways that mediate genomic stability

Genome instability has long been known to be a hallmark of cancerous cells, but the cellular causes and consequences of such instability are still not fully understood. Mutations, translocations, DNA rearrangements, as well as chromosomal loss can all result in the loss of genomic integrity. To prevent the disruption of cellular homeostasis due to DNA damage accumulation, cells contain pathways to sense and respond to DNA damage including cell cycle checkpoints and numerous DNA repair processes, collectively known as the DNA damage response (DDR). Mutations in many of the genes involved in the DDR are linked to several diseases, including premature aging, neurodegeneration and cancer. These signaling pathways are especially critical during DNA replication when the DNA is unwound and vulnerable to processing. Here, the cell relies on the S-phase checkpoint to sense DNA damage at the sites of replication forks and to facilitate a number of downstream pathways to maintain genomic stability. These processes include blocking further origin firing, facilitating DNA repair, preventing cell cycle progression, and stabilizing stalled replication forks. Here, two genome-wide siRNA screens were employed to identify additional genes involved in genome stabilization by monitoring phosphorylation of the histone variant H2AX, an early mark of DNA damage. The first screen looked at H2AX phosphorylation that occurred simply by individual protein depletion, and the second screen used a low level of a replication inhibitor, aphidicolin, to specifically identify genes that were needed to prevent DNA damage during S-phase, potentially due to the loss of replication fork stabilization mechanisms. While the results from the second screen are still undergoing further characterization, we did discover hundreds of genes whose down-regulation led to elevated levels of H2AX phosphorylation in the absence of any external stress. From this gene set, we identified many gene networks that were significantly enriched amongst our screening hits as well as several intriguing individual genes that were chosen for follow up study. These included genes involved in mRNA processing, the pathology of Charcot-Marie-Tooth syndrome, and the histone methyl transferase protein, Set8.

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