biostudies-literatureKennedy MAU.S. Department of EnergyNew England BiolabsNCI NIH HHSNational Institutes of HealthCancer Center SupportNIHNIH HHSNIGMS NIH HHSNational Science Foundation4467-4487https://www.ebi.ac.uk/biostudies/studies/S-EPMC10201449biostudies-literatureUnknown51(9)Type IIS restriction endonucleases contain separate DNA recognition and catalytic domains and cleave their substrates at well-defined distances outside their target sequences. They are employed in biotechnology for a variety of purposes, including the creation of gene-targeting zinc finger and TAL effector nucleases and DNA synthesis applications such as Golden Gate assembly. The most thoroughly studied Type IIS enzyme, FokI, has been shown to require multimerization and engagement with multiple DNA targets for optimal cleavage activity; however, details of how it or similar enzymes forms a DNA-bound reaction complex have not been described at atomic resolution. Here we describe biochemical analyses of DNA cleavage by the Type IIS PaqCI restriction endonuclease and a series of molecular stNucleic acids researchStructures, activity and mechanism of the Type IIS restriction endonuclease PaqCI.PMC10201449S10OD021832S10 OD028581DE-AC02-05CH11231DGE 1762114P30 CA015704P30 CA015704-40R01 GM105691S10 OD026941S10 OD021832Kennedy MAMorgan RDHosford CJChen MLuyten YAAzumaya CMStoddard BLfalseStructures, activity and mechanism of the Type IIS restriction endonuclease PaqCI.Type IIS restriction endonucleases contain separate DNA recognition and catalytic domains and cleave their substrates at well-defined distances outside their target sequences. They are employed in biotechnology for a variety of purposes, including the creation of gene-targeting zinc finger and TAL effector nucleases and DNA synthesis applications such as Golden Gate assembly. The most thoroughly studied Type IIS enzyme, FokI, has been shown to require multimerization and engagement with multiple DNA targets for optimal cleavage activity; however, details of how it or similar enzymes forms a DNA-bound reaction complex have not been described at atomic resolution. Here we describe biochemical analyses of DNA cleavage by the Type IIS PaqCI restriction endonuclease and a series of molecular st2023-01-01T00:00:00Z2023 May2026-04-08T13:19:01.377Z2025-04-04T14:30:41.529ZS-EPMC102014493698787410.1093/nar/gkad228