RESUMO
RECQ1 is the most abundant of the five human RecQ helicases, but little is known about its biological significance. Recent studies indicate that RECQ1 is associated with origins of replication, suggesting a possible role in DNA replication. However, the functional role of RECQ1 at damaged or stalled replication forks is still unknown. Here, for the first time, we show that RECQ1 promotes strand exchange on synthetic stalled replication fork-mimicking structures and comparatively analyze RECQ1 with the other human RecQ helicases. RECQ1 actively unwinds the leading strand of the fork, similar to WRN, while RECQ4 and RECQ5ß can only unwind the lagging strand of the replication fork. Human replication protein A modulates the strand exchange activity of RECQ1 and shifts the equilibrium more to the unwinding mode, an effect also observed for WRN. Stable depletion of RECQ1 affects cell proliferation and renders human cells sensitive to various DNA damaging agents that directly or indirectly block DNA replication fork progression. Consequently, loss of RECQ1 activates DNA damage response signaling, leads to hyper-phosphorylation of RPA32 and activation of CHK1, indicating replication stress. Furthermore, depletion of RECQ1 leads to chromosomal condensation defects and accumulation of under-condensed chromosomes. Collectively, our observations provide a new insight into the role of RECQ1 in replication fork stabilization and its role in the DNA damage response to maintain genomic stability.
Assuntos
Replicação do DNA , RecQ Helicases/metabolismo , Linhagem Celular Tumoral , Cromossomos/metabolismo , DNA/química , DNA/metabolismo , Dano ao DNA/efeitos dos fármacos , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/farmacologia , Células HeLa , Humanos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RecQ Helicases/antagonistas & inibidores , RecQ Helicases/genética , RecQ Helicases/farmacologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Proteína de Replicação A/farmacologia , Transdução de Sinais/efeitos dos fármacos , Helicase da Síndrome de WernerRESUMO
Werner syndrome (WS) is a rare autosomal progeroid disorder caused by a mutation in the gene encoding the WRN (Werner syndrome protein), a member of the RecQ family of helicases with a role in maintaining genomic stability. Genetic association studies have previously suggested a link between WRN and susceptibility to benzene-induced hematotoxicity. To further explore the role of WRN in benzene-induced hematotoxicity, we used short hairpin RNA to silence endogenous levels of WRN in the human HL60 acute promyelocytic cell line and subsequently exposed the cells to hydroquinone (HQ). Suppression of WRN led to an accelerated cell growth rate, increased susceptibility to hydroquinone-induced cytotoxicity and genotoxicity as measured by the single-cell gel electrophoresis assay, and an enhanced DNA damage response. More specifically, loss of WRN resulted in higher levels of early apoptosis, marked by increases in relative levels of cleaved caspase-7 and cleaved poly (ADP-ribose) polymerase 1, in cells treated with HQ compared with control cells. Our data suggests that WRN plays an important role in the surveillance of and protection against DNA damage induced by HQ. This provides mechanistic support for the link between WRN and benzene-induced hematotoxicity.
Assuntos
Benzeno/metabolismo , Dano ao DNA/efeitos dos fármacos , Exodesoxirribonucleases/farmacologia , Hidroquinonas/antagonistas & inibidores , Hidroquinonas/toxicidade , RecQ Helicases/farmacologia , Síndrome de Werner/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Caspase 7/metabolismo , Proliferação de Células/efeitos dos fármacos , Ensaio Cometa , Relação Dose-Resposta a Droga , Células HL-60 , Humanos , Conformação Molecular , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , RNA/química , Retroviridae/genética , Helicase da Síndrome de WernerRESUMO
The Werner syndrome protein (WRN) is mutated in Werner syndrome (WS) and plays a role in telomere maintenance, DNA repair and transcription. WS represents a premature aging syndrome with severe growth retardation. Here we show that WRN is critically required to mediate the stimulatory effect of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF-b) and epidermal growth factor (EGF) on the activity of RNA polymerase I (Pol I). Recombinant WRN specifically reconstitutes RNA polymerase I transcription in extracts from Werner syndrome fibroblasts in vitro. In addition, we identified a critical role for WRN during promoter clearance of Pol I transcription, but not in elongation. Notably, WRN was isolated in a complex with Pol I and was crosslinked to the unmethylated, active proportion of rDNA genes in quiescent cells suggesting a so far unknown role for WRN in epigenetic regulation. This together with alterations in Pol I transcription provide a novel mechanism possibly underlying at least in part the severe growth retardation and premature aging in Werner syndrome patients.
