GEN1 is a structure-specific endonuclease that resolves Holliday junctions (HJs)—four-way DNA intermediates formed during homologous recombination and DNA repair—by introducing symmetrically related cuts to produce ligatable nicked duplex products 1. The enzyme functions as a largely monomeric protein that dimerizes on HJ substrates, with first-strand cleavage being rate-limiting and second-strand cleavage occurring rapidly 1. Beyond HJ resolution, GEN1 exhibits endonuclease activity on 5'-flap and replication fork substrates 2, and recently was shown to promote common fragile site (CFS) expression by cleaving under-replicated DNA, indicating a dual role in resolving both recombination and replication intermediates 3. GEN1 contains a unique chr2 that directly contacts DNA and is essential for catalytic activity, distinguishing it within the Rad2/XPG nuclease family 1. Functionally, GEN1 is required for cell viability in the absence of other HJ resolvase activities, operating redundantly with SLX4-associated nucleases 2. Clinically, rare deleterious GEN1 variants are associated with congenital anomalies of the kidney and urinary tract (CAKUT) through impaired protein stability and enzymatic activity 4, and GEN1 variants show nominal evidence of association with aggressive prostate cancer 5.