Graduate Student - Ph.D

PRoteomics

International Centre for Genetic Engineering and Biotechnology (Trieste, Italy)

Alessandro Vindigni

Analysis of human RECQ1 helicase function in cells

RecQ helicases are a family of DNA unwinding enzymes essential for genome maintenance in all living organisms. In humans, loss of RecQ helicase function is linked with predisposition to cancer and premature aging. The present study focuses on cellular and molecular events triggered upon RECQ1 down regulation in human transformed fibroblasts (GM00637) and glioblastoma (T98G) cells. Upon acute depletion of RECQ1 by RNAi results in perturbation of S-phase progression and a significant reduction of cellular proliferation. The reduced proliferation observed upon RECQ1 depletion is associated with an increased load of DNA lesions pointing to an important role of RECQ1 in DNA repair during DNA replication. In particular, the immunofluorescence experiments showed an increased number of γ-H2AX and RAD51 foci in RECQ1 depleted cells relative to controls. The increase in the number of RAD51 foci is, however, less pronounced compared to the increase in the number of γ-H2AX foci suggesting that double strand breaks are not the major form of damage that leads to γ-H2AX foci formation in the absence of RECQ1. Consistently, there are no significant defects in homologous recombination (HRR) repair frequency in RECQ1 depleted cells. A role of RECQ1 in SSBR is also supported by the observation that the amount of the phosphorylated forms of CHK1 and ATR kinases increases upon RECQ1 depletion. RECQ1 down regulated cells are hypersensitive to replication blocking agent treatment, supporting a role of RECQ1 in restarting of stalled replication forks. Comet assays revealed that there is high load of single strand breaks in RECQ1 down-regulated cell. Collectively, the present studies provide the first indications into the specific and unique role of the human RECQ1 helicase in SSBR which is not shared by other RecQ helicases and is probably required to preserve the integrity of replication forks and allow a faithful duplication of genome.

2003-2006 Research fellow in Avestha Gengraine Technologies Pvt ltd.