Journal of Molecular Biology
Regular articleThe McrBC endonuclease translocates DNA in a reaction dependent on GTP hydrolysis1
Introduction
A recurring theme in protein-DNA enzymology is the interaction of a protein with two DNA binding sites, which can sometimes be separated by several thousand base-pairs of DNA. Communication between two sites can occur by DNA looping or DNA translocation. Examples of DNA looping include transcriptional regulators like the Lac or λ repressors (reviewed by Matthews, 1992) and proteins involved in site-specific recombination and transposition such as the λ integrase or Mu A transposase (reviewed by Gellert & Nash, 1987). In the case of the transcriptional regulators, DNA looping interactions are thought to occur by increasing the local concentration of two interacting proteins, and therefore the distances between two sites are usually not more than 500 bp and only rarely as much as 800–1000 bp as found for the deo R repressor, the enhancer protein NRI of the Escherichia coli glnALG operon and the SfiI endonuclease Reitzer and Magasanik 1986, Amouyal et al 1989, Wentzell et al 1995. Beyond these distances, the chance that two remote sites interact decreases presumably because the local concentration of the two potentially interacting complexes becomes too low (Muller-Hill, 1998). For proteins involved in site-specific recombination and transposition, the distance between recognition sites can be many thousands of base-pairs and frequently requires supercoiling. In these cases it has been suggested that the sites are brought into close proximity by topological constrains on interwrapping of the recombining sites (reviewed by Gellert & Nash, 1987).
An alternative mode for action at a distance is DNA translocation, which is involved in a variety of processes such as replication, transcription, DNA repair and DNA restriction (reviewed by Wang and Giaever 1988, Droge 1994). In cases where proteins translocate DNA actively (in contrast to passive sliding), a nucleotide co-factor, usually ATP, is required to fuel the reaction (Lohman & Bjornson, 1996). Examples of ATP-dependent translocation are helicases like DnaB (Reha-Krantz & Hurwitz, 1978), RecBCD (Taylor & Smith, 1980) and the branch migration enzymes RuvB (Iwasaki et al., 1989) and RecG (Lloyd & Sharples, 1993), MutS (Allen et al., 1997) and type I and III restriction enzymes (Bickle & Krüger, 1993).
In the case of DNA translocation, communication between two binding sites can occur over large distances. The type III restriction and modification (R-M) enzyme EcoP15I can act on recognition sites 3.5 kb apart, and the type I Eco KI enzyme can act on recognition sites 11 kb apart Meisel et al 1992, Studier and Bandyopadhyay 1988. For type I and III restriction systems, it has been established that the protein remains bound to the recognition site while translocating non-specific DNA past itself (Bickle & Krüger, 1993). DNA cleavage is supposed to be triggered when two such translocating enzyme complexes collide Studier and Bandyopadhyay 1988, Meisel et al 1992, Meisel et al 1995.
Escherichia coli K-12 expresses three modification-dependent restriction enzymes encoded by the mcrA, mrr and mcrBC genes (reviewed by Bickle & Krüger, 1993). In contrast to classical restriction-modification enzymes which act on unmodified DNA, these systems require modified DNA. McrBC specifically recognizes DNA containing either 5-hydroxymethylcytosine, 5-methylcytosine or 4-methylcytosine preceded by a purine residue (RmC) (Raleigh & Wilson, 1986). DNA cleavage by McrBC requires at least two RmC sites that are optimally separated by 40–80 bp but can be spaced as far as 3 kb apart Sutherland et al 1992, Stewart and Raleigh 1998. DNA cleavage in vitro requires the full-length gene product of the mcrB gene McrBL, McrC, GTP and Mg2+(Sutherland et al., 1992). Optimal enzyme activity is obtained at a ratio of three to five McrBL per McrC, suggesting that DNA is cleaved by a multisubunit complex (Panne et al., 1998). The activity of McrBC is modulated by a truncated gene product, McrBs, expressed by internal in-frame translation from the mcrB gene. McrBs lacks the N-terminal 161 amino acid residues and can modulate the assembly of functional McrBLMcrC complexes by binding and sequestering the McrC subunit (Panne et al., 1998). The DNA binding abilities reside in the McrBL subunit (F. J. Stewart & E.A.R., unpublished results; Krüger et al., 1995) and were localized to the protein’s N terminus, since a fragment comprising the N-terminal 190 amino acid residues retains DNA binding activity (Gast et al., 1997). McrBC is unique among the family of nucleases in that it requires GTP. This GTP dependence confirmed predictions by Dila et al. (1990), who identified a GTP-binding domain in the mcrB gene, similar to that found in other GTPases. Later it was shown that McrB has a weak intrinsic GTPase activity, which can be stimulated by McrC (Pieper et al., 1997).
We have investigated the mechanism by which McrBC accomplishes communication between remote recognition sites. Our data suggest that McrBC translocates DNA while remaining bound to its recognition site. Translocation occurred in either direction from a symmetric recognition site and was fuelled by GTP hydrolysis. DNA cleavage was triggered when two translocating complexes met or when a non-specific physical block stalled DNA translocation. We integrate these observations into a model for McrBC restriction.
Section snippets
Cleavage position
McrBC cleaves methylated DNA containing two recognition (RmC) sites (Sutherland et al., 1992). Plasmids carrying one or two recognition sites for McrBC with various spacing have been constructed previously (Stewart & Raleigh, 1998). McrBL can bind substrates having a single RmC site but they are not cleaved Sutherland et al 1992, Kruger et al 1995. The analysis of substrates carrying two RmC sites showed that DNA cleavage is optimal when the separation between recognition sites is 55–103 bp and
Protein preparations
All McrB proteins employed here were purified using the IMPACT purification system (NEB) and stored in storage buffer 10 mM Tris-HCl (pH 7.5; 21 °C), 200 mM NaCl, 0.1 mM EDTA, 1 mM DTT and 50 % (v/v) glycerol as described (Panne et al., 1998). The McrC protein used in all experiments was a gift from F.J. Stewart (NEB) purified essentially as described by Sutherland et al. (1992).
Construction of the plasmids
The spacing plasmid pMC 257 was constructed as follows: pMC 63 (Stewart & Raleigh, 1998) was cut with XbaI and SmaI
Acknowledgements
We thank Fiona J. Stewart (New England Biolabs) for providing us with purified McrC and the spacing vectors pMC 0 - pMC 3105. Maria P. MacWilliams is greatfully acknowledged for providing the lac repressor. This work was supported by the Swiss National Science Foundation.
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