title: Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA.
abstract: Fundamental aspects of the biochemistry of Type III restriction endonucleases remain unresolved despite being characterized by numerous research groups in the past decades. One such feature is the subunit stoichiometry of these hetero-oligomeric enzyme complexes, which has important implications for the reaction mechanism. In this study, we present a series of results obtained by native mass spectrometry and size exclusion chromatography with multi-angle light scattering consistent with a 1:2 ratio of Res to Mod subunits in the EcoP15I, EcoPI and PstII complexes as the main holoenzyme species and a 1:1 stoichiometry of specific DNA (sDNA) binding by EcoP15I and EcoPI. Our data are also consistent with a model where ATP hydrolysis activated by recognition site binding leads to release of the enzyme from the site, dissociation from the substrate via a free DNA end and cleavage of the DNA. These results are discussed critically in the light of the published literature, aiming to resolve controversies and discuss consequences in terms of the reaction mechanism.
authors: Biomolecular & Analytical Mass Spectrometry and Center for Proteomics (CFP-CeProMa), Department of Chemistry, University of Antwerp, Antwerp 2020, Belgium and DNA-Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.
authors: Annika
authors: A
authors: Butterer
authors: Christian
authors: C
authors: Pernstich
authors: Rachel M
authors: RM
authors: Smith
authors: Frank
authors: F
authors: Sobott
authors: Mark D
authors: MD
authors: Szczelkun
authors: Júlia
authors: J
authors: Tóth
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D015254:DNA Modification Methylases / Q000737:chemistry / Q000378:metabolism*; D015263:Deoxyribonucleases, Type III Site-Specific / Q000737:chemistry* / Q000378:metabolism*; D020035:Holoenzymes / Q000378:metabolism; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism
chemical_list: D020035:Holoenzymes; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D004247:DNA; D015254:DNA Modification Methylases; C089712:DNA modification methylase EcoP15I; C053885:DNA modification methylase EcoPI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C093225:DNA restriction enzyme EcoPI; C023451:endodeoxyribonuclease EcoP15I; D015263:Deoxyribonucleases, Type III Site-Specific