title: Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes.
abstract: Production of endonucleolytic double-strand DNA breaks requires separate strand cleavage events. Although catalytic mechanisms for simple, dimeric endonucleases are known, there are many complex nuclease machines that are poorly understood. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide after convergent ATP-driven translocation. We report the 2.7-Å resolution X-ray crystal structure of a Type ISP enzyme-DNA complex, revealing that both the helicase-like ATPase and nuclease are located upstream of the direction of translocation, an observation inconsistent with simple nuclease-domain dimerization. Using single-molecule and biochemical techniques, we demonstrate that each ATPase remodels its DNA-protein complex and translocates along DNA without looping it, leading to a collision complex in which the nuclease domains are distal. Sequencing of the products of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand-nicking events combine to produce DNA scission.
authors.affiliation: Division of Biology, Indian Institute of Science Education and Research, Pune, India.
authors.forename: Mahesh K
authors.initials: MK
authors.lastname: Chand
authors.affiliation: Division of Biology, Indian Institute of Science Education and Research, Pune, India.
authors.forename: Neha
authors.initials: N
authors.lastname: Nirwan
authors.affiliation: DNA-Protein Interactions Unit, School of Biochemistry, Biomedical Sciences Building, University of Bristol, Bristol, UK.
authors.forename: Fiona M
authors.initials: FM
authors.lastname: Diffin
authors.affiliation: DNA-Protein Interactions Unit, School of Biochemistry, Biomedical Sciences Building, University of Bristol, Bristol, UK.
authors.forename: Kara
authors.initials: K
authors.lastname: van Aelst
authors.affiliation: Division of Biology, Indian Institute of Science Education and Research, Pune, India.
authors.forename: Manasi
authors.initials: M
authors.lastname: Kulkarni
authors.affiliation: DNA-Protein Interactions Unit, School of Biochemistry, Biomedical Sciences Building, University of Bristol, Bristol, UK.
authors.forename: Christian
authors.initials: C
authors.lastname: Pernstich
authors.affiliation: DNA-Protein Interactions Unit, School of Biochemistry, Biomedical Sciences Building, University of Bristol, Bristol, UK.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Szczelkun
authors.affiliation: Division of Biology, Indian Institute of Science Education and Research, Pune, India.
authors.forename: Kayarat
authors.initials: K
authors.lastname: Saikrishnan
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D053837:DNA Cleavage; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D013294:Lactococcus lactis / Q000737:chemistry* / Q000201:enzymology; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D004720:Endonucleases; D004265:DNA Helicases