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Nucleic Acids Research, 2003, Vol. 31, No. 1 DOI: 10.1093/nar/gkg069
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2003 Oxford University Press
REBASE: restriction enzymes and methyltransferases Richard J. Roberts*, Tamas Vincze, Janos Posfai and Dana Macelis New England BioLabs, Inc., 32 Tozer Road, Beverly, MA 01915, USA Received September 23, 2002; Accepted September 27, 2002
ABSTRACT
INTRODUCTION REBASE has undergone considerable growth since the 2001 NAR Database Issue (1). In addition to restriction enzymes, methyltransferases and homing endonucleases, REBASE also includes information about other types of related proteins: nicking enzymes, specificity subunits of the Type I enzymes, control proteins and methyl-directed restriction enzymes. From biochemical screening, it seemed that perhaps 20–25% of all bacterial strains possessed restriction enzymes. However, with the advent of massive DNA sequencing efforts and the large number of complete and survey sequences now available for bacterial and archaeal genome sequences, it is clear that restriction-modification (R-M) systems are much common than had once seemed likely. These potential systems are now included within REBASE. The deduced DNA methyltransferases and restriction enzymes are given names that resemble those of normal restriction enzymes (using the conventions of reference 2), but with the suffix ‘P’ added to indicate their putative status. The REBASE web site (http://rebase.neb.com/ rebase/rebase.html) provides a summary of information known about every restriction enzyme and their associated proteins— such as commercial availability, sequence data, crystal
*To whom correspondence should be addressed. Tel: þ1 978 927 3382; Fax: þ1 978 921 1527; Email:
[email protected]
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REBASE contains comprehensive information about restriction enzymes, DNA methyltransferases and related proteins such as nicking enzymes, specificity subunits and control proteins. It contains published and unpublished references, recognition and cleavage sites, isoschizomers, commercial availability, crystal and sequence data. Homing endonucleases are also included. REBASE contains the most complete and up-to-date information about the methylation sensitivity of restriction endonucleases. In addition, there is extensive information about the known and putative restriction-modification (R-M) systems in more than 100 sequenced bacterial and archaeal genomes. The data is available on the web (http://rebase.neb.com/rebase/rebase.html), through ftp (ftp.neb.com) and as monthly updates via email.
structures, cleavage sites, recognition sequences, isoschizomers, growth temperatures and methylation sensitivity. A major focus is now on the genes that encode restriction systems and we provide both schematic illustrations of the organization of these systems and their nearest neighbours. We also provide tools (REBASE tools) that are useful in conjunction with restriction enzymes and BLAST searches can be run against all known restriction enzyme and methylase genes from the home page. There are currently 3576 biochemically-characterized restriction enzymes in REBASE. These include twelve new Type II specificities discovered since the last review (1). Of the 3516 Type II restriction enzymes, 588 are commercially available, including 211 distinct specificities from a total of 240 total specificities known. In addition, 15 DNA methyltransferases, 5 homing endonucleases and 3 nicking enzymes are commercially available. From sequence analysis of Genbank entries and other web sites such as JGI-DOE, TIGR and the Sanger Institute, there are 1411 putative genes that could be components of R-M systems. We currently have 6838 references in REBASE (journal and book publications, patents, and unpublished observations). These are complete with abstracts and with full text links, when available. References are provided for every enzyme and each fact about that enzyme is documented. REBASE has its own dedicated web server (http:// rebase.neb.com/rebase/rebase.html) and can be searched extensively. From the REBASE Lists icon on the home page, a number of tables of specialized information can be accessed. This include crystal data, cloned/sequenced genes, enzymes listed by cleavage properties and other useful compilations. Suggestions for new lists are always welcomed. An extensive effort has gone into checking and recompiling information about the sensitivity of restriction enzymes to methylation. Previous compilations (3,4) had numerous errors and each item now listed within REBASE has been checked rigorously for its accuracy. In the case of unpublished observations from those earlier compilations, individual authors have been contacted to verify the observations. In addition, all published literature has been scanned and much new information is now available. These data can be accessed both from an enzyme’s main page as well as from the ‘REBASE Methylation Sensitivity’ icon on the home page. Importantly, the data is shown in double-strand format so that the effects of hemi-methylation and doublestrand methylation are clearly differentiated.
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Figure 1. This web page shows a linear listing of known and putative R-M systems from the genome of Bacillus halodurans C-125. Methyltransferase genes are shown as thick blue arrows and restriction enzyme genes as red arrows. Flanking, non-RM genes are shown in grey. The characteristic sequence motifs fgg, pc, env, qrr, ix, x (5) or dppy, fgg (6) are shown on flags above the schematics of the methyltransferase genes. When the sequence specificity of the system is known, it is indicated at the bottom left of each schematic and the locations of the recognition sites are shown as red vertical lines. The coordinates beneath each schematic are from RefSeq (NC_002570).
The analysis of sequenced genomes has been a major focus and entries can be found from the REBASE Genomes icon. More than 120 complete or shotgun genomes have been analyzed and the results are presented in several formats. The analysis of Bacillus halodurans C-125 (GenBank # NC_002570) is shown in Figure 1. Two complete R-M systems are present, encoding BhaI (recognition sequence: GCATC) and BhaII (recognition sequence: GGCC). In
addition, an aminomethyltransferase gene is present, but none of the surrounding open reading frames show similarity to known restriction enzyme genes. This is either a solitary enzyme or the associated restriction enzyme gene is dissimilar to any known gene. The REBASE files icon brings up the growing list of currently available monthly data formats. Click on any of the numbered choices for their descriptions or to download these
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files. Click on the SUBSCRIBE TO REBASE icon to receive monthly email updates. Users who prefer retrieving REBASE data via anonymous FTP may continue to do so at ftp.neb.com (cd/pub/rebase). We also continue to maintain a monthly emailing list.
ACKNOWLEDGEMENTS Special thanks are due to the many individuals who have so kindly contributed their unpublished results for inclusion in this compilation and to the REBASE users who continue to steer our efforts with their helpful comments. We are especially grateful to Karen Otto for secretarial help. This database is supported by the National Library of Medicine (LM04971).
REFERENCES 1. Roberts,R.J. and Macelis,D. (2001) REBASE—restriction enzymes and methylases. Nucleic Acids Res., 29, 268–269. 2. Smith,H.O. and Nathans,D.J. (1973) A suggested nomenclature for bacterial host modification and restriction systems and their enzymes. J. Mol. Biol., 81, 419–423. 3. McClelland,M., Nelson,M. and Raschke,E. (1994) Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases. Nucleic Acids Res., 22, 3640–3659. 4. Nelson,M., Raschke,E. and McClelland,M. (1993) Effect of site-specific methylation on restriction endonucleases and DNA modification methyltransferases. Nucleic Acids Res., 21, 3139–3154. 5. Posfai,J., Bhagwat,A.S., Posfai,G. and Roberts,R.J. (1989) Predictive motifs derived from cytosine methyltransferases. Nucleic Acids Res., 17, 2421–2435. 6. Klimasauskas,S., Timinskas,A., Menkevicius,S., Butkiene,D., Butkus,V. and Janulaitis,A.A. (1989) Sequence motifs characteristic of DNA [cytosine-N4] methylases: similarity to adenine and cytosine-C5 DNA-methylases. Nucleic Acids Res., 17, 9823–9832.
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