Human RECQ1 promotes restart of replication forks reversed by DNA topoisomerase I inhibition

Abstract

Topoisomerase I (TOP1) inhibitors are an important class of anticancer drugs. The cytotoxicity of TOP1 inhibitors can be modulated by replication fork reversal through a process that requires poly(ADP-ribose) polymerase (PARP) activity. Whether regressed forks can efficiently restart and what factors are required to restart fork progression after fork reversal are still unknown. We have combined biochemical and EM approaches with single-molecule DNA fiber analysis to identify a key role for human RECQ1 helicase in replication fork restart after TOP1 inhibition that is not shared by other human RecQ proteins. We show that the poly(ADP-ribosyl)ation activity of PARP1 stabilizes forks in the regressed state by limiting their restart by RECQ1. These studies provide new mechanistic insights into the roles of RECQ1 and PARP in DNA replication and offer molecular perspectives to potentiate chemotherapeutic regimens based on TOP1 inhibition.

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Figure 1: Analysis of the RECQ1-PARP1 interaction.
Figure 2: Inhibition of in vitro fork restoration activity of RECQ1 by PARylated PARP1 and PAR.
Figure 3: Replication fork progression after TOP1 and PARP inhibition is impaired in RECQ1-depleted U-2 OS cells.
Figure 4: Genetic complementation with wild-type RECQ1 rescues fork progression phenotype in RECQ1-depleted cells.
Figure 5: PARP inactivation leads to DSB formation at low CPT doses in the presence but not the absence of RECQ1.
Figure 6: Reversed forks accumulate and are unable to restart in RECQ1-depleted cells after CPT treatment.
Figure 7: Schematic model of the combined roles of PARP1 and RECQ1 in response to TOP1 inhibition.

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Acknowledgements

We are grateful to A. Mazin for sharing information regarding the substrate preparation. We thank P. Janscak (University of Zurich) and D. Orren (University of Kentucky College of Medicine) for providing aliquots of purified WRN and WRN-E84A proteins, respectively. We thank G. de Murcia (École Supérieure de Biotechnologie de Strasbourg) for providing the constructs for the production of the PARP1 fragment. We thank Y. Ayala for critical discussions and G. Triolo for help in recombinant protein production. We thank the Nano Research Facility of the School of Engineering and Applied Science at Washington University in St. Louis, which is part of the National Nanotechnology Infrastructure Network supported by the US National Science Foundation under grant no. ECS-0335765, for microfabrication and the use of clean-room facility. We also thank the Center for Microscopy and Image Analysis of the University of Zurich for technical assistance with EM. This work was supported by startup funding from the Doisy Department of Biochemistry and Molecular Biology at the Saint Louis University School of Medicine and the Saint Louis University Cancer Center and grants from the President's Research Fund of Saint Louis University and the Associazione Italiana per la Ricerca sul Cancro (AIRC10510) to A.V.; US National Institutes of Health grant CA77852 to R.J.M. Jr.; Swiss National Science Foundation grants PP0033-114922 and PP00P3-135292 to M.L.; and a contribution from Fonds zur Förderung des Akademischen Nachwuchses (FAN) of the Zürcher Universitätsverein (ZUNIV) to M.L. and A.R.C.

Author information

M.B. conducted the immunoprecipitation, GST-pulldown, far-western and dot-blot experiments and the in vitro fork regression and restoration studies with the synthetic DNA substrates. A.R.C. conducted PFGE and EM analysis. S.T. conducted the single-molecule DNA replication assays. S. Gomathinayagam expressed the recombinant proteins and contributed to the in vitro fork regression and restoration assays. S.K. performed the immunofluorescence experiments and contributed to the cell-survival assays. M.V. conducted the single-molecule DNA replication assays with the BLM- and WRN-depleted cells. F.O. performed the protein complex purification experiments. T.G. contributed to the design of the proteomic experiments and performed MS analysis. S. Graziano performed the cell-survival assays. R.M.-M. contributed to the production of the GST-tagged fragments used in the GST-pulldown assays. F.M. contributed to the far-western analysis. B.L. produced the RECQ1 mutants and contributed to the optimization of protocols for RECQ1 expression. V.B. induced expression of the recombinant PARP1 protein. M.G. contributed to the design and supervision of the proteomic experiments. R.A. supervised the proteomic experiments. J.M.S. and R.J.M. Jr. contributed to the establishment of the single-molecule DNA replication assays in A.V.'s lab. R.J.M. Jr. assisted A.V. in finalizing the manuscript. M.L. planned, designed and supervised the PFGE and EM experiments and assisted A.V. in finalizing the manuscript. A.V. planned and supervised the project and wrote the manuscript.

Correspondence to Massimo Lopes or Alessandro Vindigni.

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Berti, M., Ray Chaudhuri, A., Thangavel, S. et al. Human RECQ1 promotes restart of replication forks reversed by DNA topoisomerase I inhibition. Nat Struct Mol Biol 20, 347–354 (2013) doi:10.1038/nsmb.2501

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