Lamin A/C recruits ssDNA protective proteins RPA and RAD51 to stalled replication forks to maintain fork stability

Lamin A/C provides a nuclear scaffold for compartmentalization of genome function that is important for genome integrity. Lamin A/C dysfunction is associated with cancer, aging, and degenerative diseases. The mechanisms whereby lamin A/C regulates genome stability remain poorly understood. We demonstrate a crucial role for lamin A/C in DNA replication. Lamin A/C binds to nascent DNA, especially during replication stress (RS), ensuring the recruitment of replication fork protective factors RPA and RAD51. These ssDNA-binding proteins, considered the first and second responders to RS respectively, function in the stabilization, remodeling, and repair of the stalled fork to ensure proper restart and genome stability. Reduced recruitment of RPA and RAD51 upon lamin A/C depletion elicits replication fork instability (RFI) characterized by MRE11 nuclease–mediated degradation of nascent DNA, RS-induced DNA damage, and sensitivity to replication inhibitors. Importantly, unlike homologous recombination–deficient cells, RFI in lamin A/C-depleted cells is not linked to replication fork reversal. Thus, the point of entry of nucleases is not the reversed fork but regions of ssDNA generated during RS that are not protected by RPA and RAD51. Consistently, RFI in lamin A/C-depleted cells is rescued by exogenous overexpression of RPA or RAD51. These data unveil involvement of structural nuclear proteins in the protection of ssDNA from nucleases during RS by promoting recruitment of RPA and RAD51 to stalled forks. Supporting this model, we show physical interaction between RPA and lamin A/C. We suggest that RS is a major source of genomic instability in laminopathies and lamin A/C-deficient tumors. Lamin A/C provides a nuclear scaffold for compartmentalization of genome function that is important for genome integrity. Lamin A/C dysfunction is associated with cancer, aging, and degenerative diseases. The mechanisms whereby lamin A/C regulates genome stability remain poorly understood. We demonstrate a crucial role for lamin A/C in DNA replication. Lamin A/C binds to nascent DNA, especially during replication stress (RS), ensuring the recruitment of replication fork protective factors RPA and RAD51. These ssDNA-binding proteins, considered the first and second responders to RS respectively, function in the stabilization, remodeling, and repair of the stalled fork to ensure proper restart and genome stability. Reduced recruitment of RPA and RAD51 upon lamin A/C depletion elicits replication fork instability (RFI) characterized by MRE11 nuclease–mediated degradation of nascent DNA, RS-induced DNA damage, and sensitivity to replication inhibitors. Importantly, unlike homologous recombination–deficient cells, RFI in lamin A/C-depleted cells is not linked to replication fork reversal. Thus, the point of entry of nucleases is not the reversed fork but regions of ssDNA generated during RS that are not protected by RPA and RAD51. Consistently, RFI in lamin A/C-depleted cells is rescued by exogenous overexpression of RPA or RAD51. These data unveil involvement of structural nuclear proteins in the protection of ssDNA from nucleases during RS by promoting recruitment of RPA and RAD51 to stalled forks. Supporting this model, we show physical interaction between RPA and lamin A/C. We suggest that RS is a major source of genomic instability in laminopathies and lamin A/C-deficient tumors. The spatial organization of the genome, orchestrated by structural nuclear proteins, such as A-type and B-type lamins, is important for genome function and integrity. Lamins maintain nuclear architecture, a proper response to mechanical stress, chromatin organization, and genome stability (1Denais C.M. Gilbert R.M. Isermann P. McGregor A.L. te Lindert M. Weigelin B. Davidson P.M. 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Replication stress: Getting back on track.Nat. Struct. Mol. Biol. 2016; 23: 103-109Crossref PubMed Scopus (148) Google Scholar). Upon fork stalling, there is uncoupling of DNA polymerases from the minichromosome maintenance helicase complex, which generates regions of ssDNA that are rapidly coated by the heterotrimeric RPA complex (RPA70, RPA32, and RPA14 subunits) (20Byun T.S. Pacek M. Yee M.C. Walter J.C. Cimprich K.A. Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint.Genes Dev. 2005; 19: 1040-1052Crossref PubMed Scopus (555) Google Scholar). The ssDNA–RPA complex plays a key role during RS, activating the ataxia telangiectasia–mutated and Rad3-related kinase (ATR)/Chk1-dependent S-phase checkpoint, which phosphorylates among other factors, chromatin-bound RPA32 on multiple sites including S33 (21Olson E. Nievera C.J. Klimovich V. Fanning E. Wu X. RPA2 is a direct downstream target for ATR to regulate the S-phase checkpoint.J. 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Gonzalez-Suarez I. Morgado-Palacin L. Shi W. Sage J. Roti-Roti J.L. Stewart C.L. Zhang J. Gonzalo S. A dual role for A-type lamins in DNA double-strand break repair.Cell Cycle. 2011; 10: 2549-2560Crossref PubMed Scopus (87) Google Scholar, 4Gonzalez-Suarez I. Redwood A.B. Grotsky D.A. Neumann M.A. Cheng E.H. Stewart C.L. Dusso A. Gonzalo S. A new pathway that regulates 53BP1 stability implicates cathepsin L and vitamin D in DNA repair.EMBO J. 2011; 30: 3383-3396Crossref PubMed Scopus (73) Google Scholar, 5Gonzalez-Suarez I. Redwood A.B. Perkins S.M. Vermolen B. Lichtensztejin D. Grotsky D.A. Morgado-Palacin L. Gapud E.J. Sleckman B.P. Sullivan T. Sage J. Stewart C.L. Mai S. Gonzalo S. Novel roles for A-type lamins in telomere biology and the DNA damage response pathway.EMBO J. 2009; 28: 2414-2427Crossref PubMed Scopus (168) Google Scholar, 6Liu B. Wang J. Chan K.M. Tjia W.M. Deng W. Guan X. Huang J.D. Li K.M. Chau P.Y. Chen D.J. Pei D. Pendas A.M. Cadiñanos J. López-Otín C. Tse H.F. et al.Genomic instability in laminopathy-based premature aging.Nat. Med. 2005; 11: 780-785Crossref PubMed Scopus (475) Google Scholar, 7Ghosh S. Liu B. Wang Y. Hao Q. Zhou Z. Lamin A is an endogenous SIRT6 activator and promotes SIRT6-mediated DNA repair.Cell Rep. 2015; 13: 1396-1406Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 8Liu B. Wang Z. Zhang L. Ghosh S. Zheng H. Zhou Z. Depleting the methyltransferase Suv39h1 improves DNA repair and extends lifespan in a progeria mouse model.Nat. Commun. 2013; 4: 1868Crossref PubMed Scopus (88) Google Scholar, 9Cao K. Blair C.D. Faddah D.A. Kieckhaefer J.E. Olive M. Erdos M.R. Nabel E.G. Collins F.S. Progerin and telomere dysfunction collaborate to trigger cellular senescence in normal human fibroblasts.J. Clin. Invest. 2011; 121: 2833-2844Crossref PubMed Scopus (213) Google Scholar), we determined if lamin A/C plays a role in DNA replication. Here, we show for the first time that lamin A/C is essential for the efficient recruitment of RPA and RAD51 to stalled RFs during RS and that their reduced recruitment upon lamin A/C loss causes replication fork instability (RFI) to the same extent as HR-deficient cells. Lamin A/C physically interacts with RPA (this study) and RAD51 (42Li B.X. Chen J. Chao B. Zheng Y. Xiao X. A lamin-binding ligand inhibits homologous recombination repair of DNA double-strand breaks.ACS Cent. Sci. 2018; 4: 1201-1210Crossref PubMed Scopus (11) Google Scholar) and is likely acting to recruit both RPA and RAD51 to RFs. Accordingly, lamin A/C depletion elicits nuclease-mediated degradation of stalled forks, RS-induced genomic instability, and increased sensitivity to drugs that inhibit replication. Overall, our studies indicate that reduced expression of lamin A/C, which is associated with poor prognosis in many cancers, elicits phenotypes of genomic instability because of degradation of ssDNA during replication. DNA lesions caused by endogenous and exogenous agents, secondary DNA structures, and transcribing RNA polymerases pose a challenge for the RF. Damaged RFs stall and recruit factors that protect them from excessive nucleolytic degradation, while facilitating fork restart to preserve genome stability (11Berti M. Vindigni A. Replication stress: Getting back on track.Nat. Struct. Mol. Biol. 2016; 23: 103-109Crossref PubMed Scopus (148) Google Scholar, 25Bhat K.P. Cortez D. RPA and RAD51: Fork reversal, fork protection, and genome stability.Nat. Struct. Mol. Biol. 2018; 25: 446-453Crossref PubMed Scopus (133) Google Scholar, 31Quinet A. Lemacon D. Vindigni A. Replication fork reversal: Players and guardians.Mol. Cell. 2017; 68: 830-833Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). To determine how lamin A/C loss impacts RF progression and stability upon stalling, we performed DNA fiber assays in human embryonic kidney 293T (HEK-293T) and MCF-7 cells transduced with shRNAs targeting lamin A/C (shLmna) or luciferase (shLuc) as control (Fig. 1A). Replication events were labeled with thymidine (Thy) analogs: IdU (iododeoxyuridine/red tract) followed by CldU (chlorodeoxyuridine/green tract) or vice versa (Fig. S1), for an equal amount of time and detected by immunofluorescence (IF) (43Nieminuszczy J. Schwab R.A. Niedzwiedz W. The DNA fibre technique - tracking helicases at work.Methods. 2016; 108: 92-98Crossref PubMed Scopus (50) Google Scholar). Progressing forks were identified by continuous IdU–CldU label, and the length of red (IdU) and green (CldU) tracts was measured. We find that lamin A/C depletion does not affect RF progression, as shown by the similar length of red and green tracts (Fig. S1) and an average CldU/IdU ratio of ∼1 (Fig. 1B). However, treatment with 4 mM hydroxyurea (HU) for 3 h to slow down/stall RFs results in shortening of the most recently synthesized DNA filament (CldU tract) and therefore decreased CldU/IdU ratio in lamin A/C-depleted cells (Fig. 1B). Thus, lamin A/C depletion hinders the stability of stalled RFs. Failure to protect stalled RFs causes nucleolytic degradation, a typical phenotype displayed by cells deficient in factors that promote loading of RAD51 on ssDNA like BRCA1/2 and FANCD2, cells lacking RAD51 mediators (RAD51C and XRCC2), and cells with partial inhibition of RAD51 activity or expression (24Hashimoto Y. Ray Chaudhuri A. Lopes M. Costanzo V. Rad51 protects nascent DNA from Mre11-dependent degradation and promotes continuous DNA synthesis.Nat. Struct. Mol. Biol. 2010; 17: 1305-1311Crossref PubMed Scopus (329) Google Scholar, 26Schlacher K. Christ N. Siaud N. Egashira A. Wu H. Jasin M. Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11.Cell. 2011; 145: 529-542Abstract Full Text Full Text PDF PubMed Scopus (734) Google Scholar, 33Mijic S. Zellweger R. Chappidi N. Berti M. Jacobs K. Mutreja K. Ursich S. Ray Chaudhuri A. Nussenzweig A. Janscak P. Lopes M. Replication fork reversal triggers fork degradation in BRCA2-defective cells.Nat. Commun. 2017; 8: 859Crossref PubMed Scopus (157) Google Scholar, 44Ying S. Hamdy F.C. Helleday T. Mre11-dependent degradation of stalled DNA replication forks is prevented by BRCA2 and PARP1.Cancer Res. 2012; 72: 2814-2821Crossref PubMed Scopus (220) Google Scholar). In all these contexts, there is inefficient nucleation and/or stabilization of RAD51 protofilaments, and DNA is exposed to the action of nucleases, such as MRE11, EXO1, and DNA2 (36Mason J.M. Chan Y.L. Weichselbaum R.W. Bishop D.K. Non-enzymatic roles of human RAD51 at stalled replication forks.Nat. Commun. 2019; 10: 4410Crossref PubMed Scopus (44) Google Scholar). Since MRE11 initiates end resection, whereas EXO1 and DNA2 are involved in later steps of long-range degradation (45Delamarre A. Barthe A. de la Roche Saint-André C. Luciano P. Forey R. Padioleau I. Skrzypczak M. Ginalski K. Géli V. Pasero P. Lengronne A. MRX increases chromatin accessibility at stalled replication forks to promote nascent DNA resection and Cohesin loading.Mol. Cell. 2020; 77: 395-410Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), we sought to determine whether MRE11 is responsible for fork degradation in our cells. We find that green tract shortening upon fork stalling in lamin A/C-depleted cells is due to MRE11-mediated degradation, as treatment with Mirin, an inhibitor of MRE11 nuclease, rescues fork resection (Fig. 1B). Consistently, MRE11 depletion by siRNA restores the average CldU/IdU ratio to ∼1 in lamin A/C-depleted cells (Fig. 1C). Importantly, lamin A/C loss does not affect cell cycle progression, as indicated by the normal cell cycle profile observed by flow cytometry (Fig. S2). Similarly, the 3-h treatment with HU and Mirin does not alter the percentage of cells in S phase (Fig. S2). These control experiments support the validity of our cellular model and experimental conditions to study DNA replication. Interestingly, the decreased CldU/IdU ratio upon RF stalling and the rescue of replication defects by Mirin were also observed in lamin A/C-depleted tumor cells—MCF-7 (Fig. 1D) and U2OS (Fig. S3), and in mouse embryonic fibroblasts (MEFs) from Lmna−/− mice (Fig. 1E). These data indicate that lamin A/C is required for the stability of RFs in multiple cell types. Moreover, compounds such as camptothecin and aphidicolin (APH), which cause RF stalling via different mechanisms, also elicit RFI in lamin A/C-depleted cells (Fig. 1F). Importantly, reconstitution of lamin A in lamin A/C-depleted cells rescues replication defects upon HU treatment (Fig. 1G). In contrast, expression of a truncated mutant form of lamin A (LAΔ50), also known as "progerin," does not rescue replication defects. Rather, progerin itself imposes a challenge to DNA replication, causing RFI (reduced CldU/IdU ratio) in the absence of any drug. This confirms our published results showing fork stalling and nuclease-mediated degradation of newly replicated DNA upon expression of progerin in normal cells (46Kreienkamp R. Graziano S. Coll-Bonfill N. Bedia-Diaz G. Cybulla E. Vindigni A. Dorsett D. Kubben N. Batista L.F.Z. Gonzalo S. 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