Structure-based sequence alignment of type-II restriction endonucleases

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Abstract

The type-II restriction endonucleases generally do not share appreciable amino acid sequence homology. The crystal structures of restriction endonucleases EcoRI and BamHI have shown these enzymes to possess striking 3D-structural resemblance, i.e., they have a similar overall fold and similar active sites, though they possess <23% sequence identity. Structural superimposition of EcoRI, BamHI, EcoRV, and PvuII based on active site residues led to sequence alignments which showed nine possible sequence motifs. EcoRV and PvuII show a more similar pattern than EcoRI and BamHI suggesting that they belong to a different subgroup. The motifs are characterized by charged and/or hydrophobic residues. From other studies on the structure of these endonucleases, three of the motifs could be implicated in DNA binding, three in forming the active site and one in dimer formation. However, the motifs were not identifiable by regular sequence alignment methods. It is found that motif IX in BamHI is formed by reverse sequence order and the motif IX in PvuII is formed from the symmetry related monomer of the dimer. The inter-motif distance is also quite different in these cases. Of the nine motifs, motif III has been earlier identified as containing the PD motif involving one of the active site residues. These motifs were used in a modified profile analysis procedure to identify similar regions in eight other endonuclease sequences for which structures are not known.

Introduction

Restriction-modification systems in bacteria have two functional components, the DNA restriction activity and the DNA methylase activity, both recognizing the same sequence. The restriction enzymes in bacteria recognize and cleave foreign DNA that is unmodified (unmethylated). The type-II restriction-modification system consists of different enzymes for methylation and restriction, while type I and III systems have both the methylating and restricting activities present in the same enzyme. Type-II restriction endonucleases cleave double stranded DNA in the presence of Mg2+ ions with high specificity at sites near or within the recognition sequences. (for review see [1], [2]). Type-IIs restriction endonucleases recognize asymmetric sites, 4–7 bp long and cleave in presence of Mg2+ with a staggered cut, at a fixed distance from one side of recognition site. These are also larger than type-IIp enzymes and may contain two or more domains, a recognition domain and a cleaving domain [3]. Type-IIe enzymes have a two site DNA cleavage mechanism and resemble proteins involved in transcriptional regulation, DNA recombination and replication [4], [5], [6].

The type-II restriction endonucleases lack appreciable sequence identity, (<23%) (Table 1), in contrast to DNA methylases that do have some conserved motifs of amino acid sequence [7]. Hence, the restriction endonucleases have been thought to be unrelated to each other. Sequence alignment of endonucleases in conjunction with phenotypic recognition sequence data has been used in an earlier report to cluster the endonucleases [8].

X-ray crystallographic studies on type-II restriction endonucleases have shown commonality in their structures. The structures of restriction endonucleases BamHI [9], EcoRI [10], [11], EcoRV [12] PvuII [13], Cfr10I [14], BglI [15] and MunI [16] have been determined and studies on the structural and functional aspects have been made [17], [18], [19], [20], [21], [22]. The structural topology is comparable (Fig. 1). The structures of BamHI and EcoRI show a striking presence of a common structural fold comprising a central bundle of five β-strands and four α-helices on the sides [23]. Comparison of the structures of EcoRI and EcoRV showed that there is a common structural motif containing two acidic side chains and one basic side chain near the scissile phosphodiester bond [12]. This motif called the PD…(active site) motif was also predicted to be present in a few other type-II restriction endonucleases [1]. Homologs amongst other restriction enzymes could not be detected by Newman et al. [24] using profile-based search methods based on structurally identified common core region between EcoRI and BamHI.

Different sequences can give rise to very similar folds [25]. Techniques for superimposition of protein structures have been developed [26]. Superimposing such similarly folded protein structures belonging to same functional class and allowing appropriate insertions or deletions in the sequences based on the structural alignment, it has been possible to find conserved motifs. Structure based sequence alignment has been reported for a variety of systems, for example, between Mengo virus and HRV14 [27], DNA methyltransferases [28], [29], replication terminator protein from Bacillus subtilis [30] and zinc endopeptidases [31].

We report here the structure based sequence alignment of the type-II restriction endonucleases EcoRI, BamHI, EcoRV, and PvuII and the detection of nine possible motifs. We have extended the analysis to a few other type-IIp endonucleases sequences using a profile search method.

Section snippets

Materials and methods

The structures of EcoRI (1ERI), BamHI (1BAM), EcoRV (4RVE) and PvuII (2PVI) were from the Protein Data Bank [32]. The set of sequences of restriction endonucleases for which structures are not known comprises representative type-IIp sequences with different substrate specificities. Type-IIs endonucleases were not used for analysis as they have a two-domain structure. EcoRII, a representative of type-IIe enzymes was removed from the analysis as it is shown to be presumably a dimer of

Structural and sequence alignment of restriction endonucleases EcoRI, BamHI, EcoRV and PvuII

Structural alignment of crystallographic structures of EcoRI and BamHI show them to share a common structural fold as reported earlier. Alignment of crystallographic structures of restriction endonucleases EcoRV and PvuII based on their active site residues with the structures of EcoRI and BamHI showed that these two enzymes also share a part of the common structural fold.

The sequences aligned on the basis of structural identity led to the identification of nine motifs in amino acid sequences

Discussion

We used a profile search method [34] with different profile combinations to identify a set of nine motifs (five strong, four weak), and these motifs were validated from the alignment of EcoRI, BamHI, EcoRV and PvuII structures. The motif IX found in BamHI is a reverse motif i.e., it is the sequence found in the N-terminal region of the BamHI sequence, comprising residues 32–19 which are structurally similar to the region corresponding to residues 274–287 in EcoRI. There are examples where the

Acknowledgements

We thank the Department of Biotechnology, Government of India, for the Bioinformatics Centre facilities. T.D. thanks CSIR for the fellowship. We thank the anonymous reviewer for pointing out that EcoRII is a type-IIe enzyme and showing the BglII structure.

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