Journal of Molecular Biology
Volume 272, Issue 2, 19 September 1997, Pages 190-199
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Regular article
Characterization of the interaction between the restriction endonuclease McrBC from E. coli and its cofactor GTP1

https://doi.org/10.1006/jmbi.1997.1228Get rights and content

Abstract

McrBC, a GTP-dependent restriction enzyme from E. coli K-12, cleaves DNA containing methylated cytosine residues 40 to 80 residues apart and 3′-adjacent to a purine residue (PumCN40–80PumC). The presence of the three consensus sequences characteristic for guanine nucleotide binding proteins in one of the two subunits of McrBC suggests that this subunit is responsible for GTP binding and hydrolysis. We show here that (i) McrB binds GTP with an affinity of 106 M−1 and that GTP binding stabilizes McrB against thermal denaturation. (ii) McrB binds GDP about 50-fold and ATP at least three orders of magnitude more weakly than GTP. (iii) McrB hydrolyzes GTP in the presence of Mg2+ with a steady-state rate of approximately 0.5 min−1. (iv) McrC stimulates GTP hydrolysis 30-fold, but substrate DNA has no detectable effect on the GTPase activity of McrB, neither by itself nor in the presence of McrC. (v) Substitution of N339 and N376 with alanine allowed us to identify NTAD (339 to 342) rather than NKKA (376 to 379) as the equivalent of the third consensus sequence motif characteristic for guanine nucleotide binding proteins, NKXD.

Introduction

Escherichia coli K-12 strains code for several restriction enzymes selectively directed against DNA containing modified bases (reviewed by Noyer-Weidner & Trautner, 1993). These enzymes are encoded by the mcrA, mcrBC and mrr genes, respectively Noyer-Weidner et al 1986, Raleigh and Wilson 1986, Heitman and Model 1987. mcrA is carried by the excisable prophage-like element e14, which is integrated into the chromosome at about 25 minutes on the E. coli K-12 standard map (Raleigh et al., 1989). The mcrBC and mrr genes, which flank the determinants, hsdRMS, of the “classical” type I R/M system EcoK, are located at about 98.5 minutes Noyer-Weidner et al 1986, Heitman and Model 1987, Raleigh et al 1989.

The identification of the mcrA and mcrBC genes as specifying restriction of methylated DNA was actually a rediscovery. They proved to be identical Noyer-Weidner et al 1986, Raleigh and Wilson 1986, Raleigh et al 1989 with the rglA and rglB loci, respectively, previously described in the context of restriction of non-glucosylated T-even phage DNA (Luria & Human, 1952; reviewed by Revel, 1983). Such DNA is prone to restriction by the corresponding activities as it contains 5-hydroxymethylcytosine residues.

Sequence analysis of the mcrBC region Ross et al 1989, Dila et al 1990 revealed two partly overlapping reading frames coding for major products of ca 53 and 37 kDa. Genetic studies seemed to indicate that some restriction phenotypes associated with the mcrBC locus would be mediated by the 53 kDa McrB protein alone Dila et al 1990, Kelleher and Raleigh 1991. This finding, however, could not be confirmed by subsequent in vitro experiments (Sutherland et al 1992, Kruger et al 1995; U. P., unpublished) showing the requirement for both McrB and McrC in restriction of substrates accessible by the mcrBC -specified activity.

The McrBC restriction enzyme cleaves DNA substrates carrying two PumC sequences spaced by approximately 40 to 80 non-defined base-pairs. 5-Hydroxymethylcytosine (5hmC), 5-methylcytosine (5 mC) and N -4-methylcytosine (N4 mC) residues, in the constellation described, permit restriction. Cleavage occurs in between the two PumC sites and requires GTP and Mg2+ (Sutherland et al., 1992). It is the strict requirement for GTP hydrolysis which makes McrBC unique among restriction enzymes (for reviews see: Roberts and Halford 1993, Pingoud and Jeltsch 1997). The sequence of McrB suggests that the GTP binding site is located in the C-terminal half of the molecule, where three consensus sequences characteristic for guanine nucleotide binding proteins (Dever et al., 1987) are located (Sutherland et al., 1992): GPPGVGKT (position 207 to 214), DKRG (306 to 309), and NTAD (339 to 342) (or, alternatively, NKKA 376 to 379, vide infra). While this suggests that McrB is responsible for GTP binding and hydrolysis, this has not yet been demonstrated experimentally. McrB also harbors the DNA binding site, as it was shown that McrB, stimulated by but not dependent on GTP and McrC, specifically binds DNA with McrBC recognition sites, whereas unmethylated DNA is bound very weakly (Krüger et al., 1995).

In an effort to understand the requirement of the restriction activity for GTP hydrolysis we have begun to analyze the interaction of the McrBC system with DNA and guanine nucleotides. Here we present our results concerning GTP binding and hydrolysis by McrB and selected mutants in the absence and presence of McrC. We demonstrate that McrB is indeed the component of the McrBC restriction enzyme which is responsible for GTP binding and hydrolysis. It binds GTP with an affinity of 106 M−1, about 50 times more strongly than GDP. It hydrolyzes GTP with an intrinsic rate of approximately 0.5 min−1, which is stimulated 30-fold by McrC, but not by DNA. Intriguingly, the steady-state GTP hydrolysis rate is much faster than the steady-state DNA hydrolysis rate, suggesting that many turnovers of GTP hydrolysis are taking place before DNA cleavage occurs.

Section snippets

McrB is the GTP-binding and hydrolyzing subunit of the McrBC restriction system

In order to investigate the role of the two components of the McrBC restriction system regarding GTP binding and hydrolysis, we assayed GTP hydrolysis by McrB and McrC alone or in combination. McrB slowly hydrolyzes GTP in a steady-state manner, whereas McrC alone has no GTPase activity. Addition of McrC to McrB results in a considerable increase of GTPase activity (Figure 1). Figure 2 shows the McrC concentration dependence of the GTPase-stimulating effect on McrB at two different McrB

Discussion

Restriction of methylated DNA by McrBC is strictly GTP-dependent. Based on the amino acid sequence it was assumed by Sutherland et al. (1992) that McrB is the GTP binding entity of the McrBC restriction system. McrB contains the tripartite sequence motif characteristic for GTP binding proteins, namely GPPGVGKT (position 207 to 214), DKRG (306 to 309), and NTAD (339 to 342), which led to the conclusion that McrB is a member of the GTPase superfamily. The first, albeit indirect, experimental

Bacterial strains, plasmids and media

For the expression of McrB and McrC as well as for the preparation of methylated substrate plasmids, the methylation tolerant E. coli strain TC410 [mcrA, Δ(mrr-hsdRMS-McrBC)201, minA, minB, rpsL, sup+] Noyer-Weidner et al 1986, Kruger et al 1992 was used. McrB and McrC were expressed as GST-fusion proteins encoded by the plasmids pBN211 and pBN213, respectively Kruger 1992, Kruger et al 1995. E. coli cells were grown in LB medium with 50 μg/ml ampicillin.

Enzymes, chemicals and radiochemicals

Human plasma thrombin was purchased

Acknowledgements

We thank U. Steindorf for expert technical assistance and Dr F.-U. Gast for critical reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (Pi 122/11-1) and the Fonds der Chemischen Industrie.

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    Present address: T. Krüger and M. Noyer-Weidner, Walter de Gruyter & Co, Scientific Publishers, Genthiner Str. 13, D-10728 Berlin, Germany.

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