<p>SIRT1 (silent mating type information regulation 2 homolog 1) functions as a versatile regulatory protein that modulates metabolism, epigenetic states, and DNA repair, thereby playing a critical role in maintaining cellular homeostasis. As a major protein deacetylase, SIRT1 acts on multiple substrates, with RECQL4 being a prominent DNA repair target. SIRT1-mediated deacetylation of RECQL4 regulates DNA replication and base excision repair, both of which are essential for genome maintenance. Genetic variants in SIRT1 have been associated with diminished deacetylase activity and many clinical ailments, including cancer. Non-synonymous single-nucleotide polymorphisms (nsSNPs) can impact the structure and function of the SIRT1 protein and impair its interactions with target proteins. Therefore, this study aimed to identify detrimental nsSNPs in SIRT1 and evaluate their potential impact on the SIRT1-RECQL4 interaction using an in-silico framework. The stepwise pathogenicity prediction of SNPs using advanced computational tools predicted five nsSNPs (R282C, R341C, P323S, D483Y, and P447S) as most damaging and highly deleterious. Subsequent docking analysis of these five SIRT1 variants with RECQL4 revealed that the D483Y mutation was the most functionally deleterious. To assess the effects of the D483Y variant on SIRT1 protein conformation and its dynamic interaction with RECQL4, 500 ns long molecular dynamics simulations (MDS) were conducted for both the wild-type and mutant SIRT1-RECQL4 complexes. MDS results indicated that the D483Y mutation reduces conformational flexibility in the otherwise dynamic SIRT1-RECQL4 complex. These findings offer structural evidence of how single amino acid substitutions can compromise SIRT1 function and provide new mechanistic insights into the SIRT1-RECQL4 regulatory axis in oncogenic progression.</p> Graphical Abstract <p></p>