Abstract The spatial organization of the genome has emerged as an additional level of regulation of genome function and integrity. Lamins provide a scaffold for the compartmentalization of genome functions, being important for nuclear architecture, response to mechanical stress, chromatin organization, and DNA transcription, replication and repair. These findings, and the association of lamins mutations with degenerative disorders, premature aging, and cancer, provide evidence for these proteins operating as “caretakers of the genome”. However, the mechanisms whereby lamins regulate genome function and stability remain poorly understood. Unveiling these mechanisms is key to identify therapies that ameliorate the progression of lamin-related diseases in patients. Our proposal combines molecular, cellular, and organismal studies, to identify new mechanisms contributing to the pathology of laminopathies, focusing on Hutchinson Gilford Progeria Syndrome (HGPS), a premature aging disease caused by a mutant lamin A protein called “progerin”. We present evidence for lamins playing a direct role in DNA replication. Lamins depletion reduces recruitment of factors that protect stalled forks, leading to nuclease-mediated fork degradation, and replication stress (RS)-induced genomic instability. Progerin expression elicits a more robust effect on DNA replication, causing replication fork stalling, in addition to fork deprotection and degradation. RS in progerin-expressing cells is accompanied by upregulation of the cGAS/ STING cytosolic DNA sensing pathway, and activation of a STAT1-regulated IFN-like response. This response has received much attention lately due to its involvement in malignant transformation and senescence/aging. Importantly, treatments that ameliorate HGPS cellular phenotypes, especially calcitriol, reduces RS, represses the IFN-like response, and increases reprogramming efficiency, a paradigm of rejuvenation. Here, we will use new technologies such as single-molecule replication assays (DNA fibers), iPOND (Isolation of Proteins On Nascent DNA) and electron microscopy to identify molecular mechanisms whereby lamins loss and progerin expression hinder DNA replication (Aim 1). In addition, we will determine the cause-and-effect relationship between RS and activation of the cGAS/STING pathway and the STAT1-regulated IFN-like response, and the consequences of these alterations for cellular fitness (Aim 2). Moreover, we will test whether the broad beneficial effects of calcitriol in cells in vitro translate into amelioration of phenotypes in vivo using mouse models of laminopathies (Aim 3). If successful, our study will fill gaps in our knowledge about mechanisms whereby lamins ensure proper DNA replication, advance laminopathies’ research by identifying new pathways contributing to cellular and organismal deterioration, and provide evidence for the benefits of calcitriol in preclinical models, which will serve as proof-of-concept for its utili...