Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Fukuyo, M., T. Nakano, Y. Zhang, Y. Furuta, K. Ishikawa, M. Watanabe-Matsui, H. Yano, T. Hamakawa, H. Ide, and I. Kobayashi. “Restriction-Modification System with Methyl-Inhibited Base Excision and Abasic-Site Cleavage Activities.” Nucleic Acids Research 43, no. 5 (February 19, 2015): 2841–2852. As Published http://dx.doi.org/10.1093/nar/gkv116 Publisher Oxford University Press Version Final published version Citable link http://hdl.handle.net/1721.1/98182 Terms of Use Creative Commons Attribution Detailed Terms http://creativecommons.org/licenses/by/4.0/ Published online 19 February 2015 Nucleic Acids Research, 2015, Vol. 43, No. 5 2841–2852 doi: 10.1093/nar/gkv116 Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities Masaki Fukuyo1,2,3,4,†, Toshiaki Nakano5,†, Yingbiao Zhang1,2, Yoshikazu Furuta1,2, Ken Ishikawa1,2,6, Miki Watanabe-Matsui1,2, Hirokazu Yano1,2, Takeshi Hamakawa5, Hiroshi Ide5 and Ichizo Kobayashi1,2,6,* 1Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, Tokyo 108-8639, Japan, 2Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan, 3Department of Evolutionary Studies of Biosystems, School of Advanced Sciences, The Graduate University for Advanced Studies (SOKENDAI), Hayama, Kanagawa 240-0193, Japan, 4Department of Molecular Oncology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan, 5Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University Higashi-Hiroshima 739-8526, Japan and 6Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Tokyo 113-8654, Japan Received June 10, 2014; Revised February 04, 2015; Accepted February 05, 2015 ABSTRACT understanding of genetic and epigenetic processes linking those in prokaryotes and eukaryotes. The restriction-modification systems use epigenetic modification to distinguish between self and non- self DNA. A modification enzyme transfers a methyl INTRODUCTION group to a base in a specific DNA sequence while Restriction-modification (RM) systems recognize and at- its cognate restriction enzyme introduces breaks in tack nonself DNA based on DNA chemical modifications DNA lacking this methyl group. So far, all the restric- (Figure 1A). Among the various types of chemical mod- tion enzymes hydrolyze phosphodiester bonds link- ification, epigenetic methylation of bases is well studied. ing the monomer units of DNA. We recently reported Methylation occurs at specific bases, generating m5C (5- that a restriction enzyme (R.PabI) of the PabI super- methylcytosine, 5mC), m4C (N4-methylcytosine) or m6A family with half-pipe fold has DNA glycosylase activ- (N6-methyladenine, mA) at specific sequences (1). RM sys- ity that excises an adenine base in the recognition tems are frequently encountered in the prokaryotic world and, less often, in the eukaryotic world (REBASE, http: sequence (5 -GTAC). We now found a second activity / //rebase.neb.com)(2,3). These systems show mobility and in this enzyme: at the resulting apurinic apyrimidinic variability in sequence recognition (4) and interact in co- = (AP) (abasic) site (5 -GT#C, # AP), its AP lyase activ- operative or conflicting ways (5). RM systems have regula- ity generates an atypical strand break. Although the tory mechanisms reminiscent of mobile elements and toxin– lyase activity is weak and lacks sequence specificity, antitoxin systems (6). Bacterial strains can have very differ- its covalent DNA–R.PabI reaction intermediates can ent RM systems and methylomes, even genomic informa- be trapped by NaBH4 reduction. The base excision is tion indicates that they are closely related (7). not coupled with the strand breakage and yet causes The biological significance of RM systems is not yet fully restriction because the restriction enzyme action can understood. RM systems were discovered through host con- impair transformation ability of unmethylated DNA trolled variation of bacterial viruses: a virus propagated in even in the absence of strand breaks in vitro. The one host might not grow well in another host because of differences in DNA modification (8). RM systems attack base excision of R.PabI is inhibited by methylation of incoming DNA such as viral genomes and transforming the target adenine base. These findings expand our DNA as well as endogenous genomes with nonself epige- *To whom correspondence should be addressed. Tel: +81 3 5449 5326; Fax: +81 3 5449 5422; Email: [email protected] †These authors contributed equally to the paper as first authors. Present addresses: Masaki Fukuyo, Department of Molecular Oncology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan. Yoshikazu Furuta, Institute for Medical Engineering & Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Ken Ishikawa, Gene Regulation and Chromosome Biology Laboratory, National Cancer Institute at Frederick, National Institutes of Health, Frederick, MD 21702, USA. Miki Watanabe-Matsui, Department of Biochemistry, School of Medicine, Tohoku University, Sendai 980-8575, Japan. C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 2842 Nucleic Acids Research, 2015, Vol. 43, No. 5 result suggested that DNA ends generated by R.PabI might be different from the 3-OH and 5-P ends. The product ends generated by purified R.PabI were difficult to religate, sug- gesting again that they did not have the typical 3-OH and 5-P end structures (Figure 1B (ii), see below). Our recent structural biology-based work demonstrated that R.PabI is a DNA glycosylase that excises an adenine base from the recognition sequence (Figure 1B (iii)) (20). This was unex- pected because base excision has often been associated with repair of DNA damages. It also reminds of excision of a Figure 1. Restriction enzymes. (A) Restriction-modification systems. For 5-methylcytosine base and its oxidized derivatives in DNA a specific DNA sequence, an enzyme makes epigenetic modifications such demethylation in plants and animals (21,22). as methylation to label self DNA. DNA without the modification is at- Finding of the base excision activity of the restriction en- tacked by the paired restriction enzyme. Duplex lines, double-stranded zyme immediately raises two questions. The first is about DNA. Box, specific sequence. Magenta diamond, chemical modification its relation to DNA strand breakage. The above work such as methylation (Me) at the recognition sequence. (B) Two possible / routes to DNA breakage for the restriction enzyme R.PabI. Top, a double- (20) concluded that the resulting AP (apurinic apyrimidinic stranded DNA with a recognition sequence (i). Left, hydrolysis of phos- = abasic) site is transformed into a strand break by the phodiester bonds to generate two 3-OH and 5-P ends (ii). Right, gener- ␤-elimination reaction at a high temperature in the (hy- ation of AP sites (iii). Cleavage generates two strand breaks (iv) with 5 -P per)thermophilic bacteria or by a separate AP endonuclease and 3-modified sugar (gray oval) ends. Red A, adenine to be excised unless methylated by a paired modification enzyme. See Supplementary Figure S4 in the mesophilic bacteria. A line of evidence for the latter for more detailed reaction mechanisms. route is the specific cleavage by the mixture of R.PabI and Escherichia coli lysate (20). This is consistent with specific cleavage with E. coli extract containing R.HpyAXII, a H. netic status (9,10). RM systems thus promote genetic isola- pylori homolog of R.PabI (19). The second question is its tion of a lineage. We hypothesize that they also drive adap- role in restriction. Which could be responsible for restric- tive evolution by introducing a specific global gene expres- tion phenomenon, base excision or strand breakage? sion pattern (7,11–12). In the present work, we addressed these questions. We All restriction enzymes examined so far hydrolyze phos- demonstrated a second activity in this enzyme that gener- phodiester bonds linking monomer nucleotide units leav- ates an atypical strand break at the AP site (Figure 1B(iv)). ing 3-OH (hydroxyl) and 5-P (phosphate) ends (Figure 1B This cleavage is, however, not always coupled to the base (i, ii)). Thus, they are phosphodiesterases. A break on one excision. The base excision reaction turned out to be suf- strand is often coupled with a nearby break on the comple- ficient for restriction because the enzyme action impaired mentary strand, leading to a double-strand break. Restric- DNA biological activity in the absence of strand breakage tion enzymes of Type II RM systems introduce a break at or in vitro. near a specific DNA sequence unless the sequence is methy- lated at a specific base (1). Type II restriction enzymes have been important for molecular biology and genetic engineer- MATERIALS AND METHODS ing (8). In addition, studies of their function and structure Bacterial strains, plasmids, viral genome and oligonucleotides