The GTP-dependent restriction enzyme McrBC from Escherichia coli forms high-molecular mass complexes with DNA and produces a cleavage pattern with a characteristic 10-base pair repeat.
The GTP-dependent restriction enzyme McrBC consists of two polypeptides: one (McrB) that is responsible for GTP binding and hydrolysis as well as DNA binding and another (McrC) that is responsible for DNA cleavage. It recognizes two methylated or hemimethylated RC sites (R(m)C) at a distance of approximately 30 to more than 2000 base pairs and cleaves the DNA close to one of the two R(m)C sites. This process is strictly coupled to GTP hydrolysis and involves the formation of high-molecular mass complexes. We show here using footprinting techniques, surface plasmon resonance, and scanning force microscopy experiments that in the absence of McrC, McrB binds to a single R(m)C site. If a second R(m)C site is present on the DNA, it is occupied independently by McrB. Whereas the DNA-binding domain of McrB forms 1:1 complexes with each R(m)C site and shows a clear footprint on both R(m)C sites, full-length McrB forms complexes with a stoichiometry of at least 4:1 at each R(m)C site, resulting in a slightly more extended footprint. In the presence of McrC, McrB forms high-molecular mass complexes of unknown stoichiometry, which are considerably larger than the complexes formed with McrB alone. In these complexes and when GTP is present, the DNA is cleaved next to one of the R(m)C sites at distances differing by one to five helical turns, suggesting that in the McrBC-DNA complex only a few topologically well-defined phosphodiester bonds of the DNA are accessible for the nucleolytic center of McrC.
Pieper
Uwe
U
Institut für Biochemie (FB 08), Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany.
Groll
Detlef H
DH
Wünsch
Sandra
S
Gast
Frank-Ulrich
FU
Speck
Christian
C
Mücke
Norbert
N
Pingoud
Alfred
A
D020029:Base Pairing; D003432:Cross-Linking Reagents / Q000378:metabolism; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D018983:DNA Footprinting; D004262:DNA Restriction Enzymes / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D046911:Macromolecular Substances; D018625:Microscopy, Atomic Force; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010777:Photochemistry; D012091:Repetitive Sequences, Nucleic Acid; D020349:Surface Plasmon Resonance
D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; C008143:5-iodocytosine; D006160:Guanosine Triphosphate; D003596:Cytosine; D004262:DNA Restriction Enzymes; C120301:McrBC endonuclease; C059678:mcrB protein, E coli