Hyperthermophilic DNA Methyltransferase M.PabI from the Archaeon Pyrococcus abyssi

Abstract

Genome sequence comparisons among multiple species of Pyrococcus, a hyperthermophilic archaeon, revealed a linkage between a putative restriction-modification gene complex and several large genome polymorphisms/rearrangements. From a region apparently inserted into the Pyrococcus abyssi genome, a hyperthermoresistant restriction enzyme [PabI; 5-(GTA/C)] with a novel structure was discovered. In the present work, the neighboring methyltransferase homologue, M.PabI, was characterized. Its N-terminal half showed high similarities to the M subunit of type I systems and a modification enzyme of an atypical type II system, M.AhdI, while its C-terminal half showed high similarity to the S subunit of type I systems. M.PabI expressed within Escherichia coli protected PabI sites from RsaI, a PabI isoschizomer. M.PabI, purified following overexpression, was shown to generate 5-GTm6AC, which provides protection against PabI digestion. M.PabI was found to be highly thermophilic; it showed methylation at 95°C and retained at least half the activity after 9 min at 95°C. This hyperthermophilicity allowed us to obtain activation energy and other thermodynamic parameters for the first time for any DNA methyltransferases. We also determined the kinetic parameters of k cat , K m, DNA , and K m, AdoMet . The activity of M.PabI was optimal at a slightly acidic pH and at an NaCl concentration of 200 to 500 mM and was inhibited by Zn 2؉ but not by Mg 2؉ , Ca 2؉ , or Mn 2؉ . These and previous results suggest that this unique methyltransferase and PabI constitute a type II restriction-modification gene complex that inserted into the P. abyssi genome relatively recently. As the most thermophilic of all the characterized DNA methyltransferases, M.PabI may help in the analysis of DNA methylation and its application to DNA engineering.DNA methylation plays a crucial role in diverse biological processes, for example, replication and repair of DNA, expression and silencing of genes, and distinction between self and nonself DNAs. DNA methyltransferases require S-adenosyl-Lmethionine (AdoMet) as the donor of a methyl group (7). The common core of the AdoMet-dependent methyltransferases is formed by seven-stranded ␤-sheets (53), and the target base within a double-stranded DNA molecule is installed into its catalytic pocket by a "base-flipping" mechanism (20). The AdoMet-dependent DNA methyltransferases are classified into three groups according to their products: those yielding 5-methylcytosine (m5C), those yielding N 4 -methylcytosine (m4C), and those yielding N 6 -methyladenine (m6A). The latter two groups of amino-nitrogen methyltransferases are further subdivided into ␣, ␤, ␥, and other families, according to the order of motifs (of the catalytic center and AdoMet-binding region) and the target recognition domain (TRD) (4, 27).Most of the prokaryotic DNA methyltransferases constitute restriction-modification (RM) systems, which can be classified into types I, II, and III (45,46). A classic type II RM system is composed of two distinct prote...

Transmission of the PabI family of restriction DNA glycosylase genes: mobility and long-term inheritance

“…R.PabI was originally identified from a hyperthermophilic archaeon Pyrococcus abyssi during genome comparison with Pyrococcus horikoshii [ 4 , 5 ]. A region including the R.PabI gene and the neighboring methyltransferase gene encoding a DNA methyltransferase, M.PabI, is present in the genome of P. abyssi , but absent at the orthologous locus in the genome of P. horikoshii [ 10 ], which contains an 18 kb sequence instead [ 5 ]. Homologs of R.PabI have been detected in various archaea and bacteria, with particular abundance in Epsilonproteobacteria, such as Helicobacter pylori [ 7 ].…”

Novel protein fold discovered in the PabI family of restriction enzymes

“…The uninduced level of R.PabI killed Escherichia coli cells even when the cognate methyltransferase M.PabI (15) was co-expressed (M.W. and I.K., unpublished data).…”

Genome-wide DNA methylation analysis of Haloferax volcanii H26 and identification of DNA methyltransferase related PD-(D/E)XK nuclease family protein HVO_A0006

“…Previous research on DNA methylation has focused primarily on eukaryotic and bacterial organisms. The few studies which have examined DNA methylation and RM systems in archaeal organisms have concentrated on a few restriction endonucleases and methyltransferases, with no emphasis on overall genomic methylation (Baranyi et al, 2000 ; Chinen et al, 2000 ; Grogan, 2003 ; Ishikawa et al, 2005 ; Kim et al, 2005 ; Watanabe et al, 2006 ). In this study, we characterized genome-wide DNA methylation patterns of an archaeal organism.…”

“…Although RM systems and DNA methylation have been extensively studied in bacteria, few studies have examined these systems in archaea. Most research on archaeal RM systems has focused on the activity of restriction endonucleases and methyltransferases in hyperthermophilic organisms, such as those belonging to the genus Pyrococcus (Chinen et al, 2000 ; Ishikawa et al, 2005 ; Watanabe et al, 2006 ). Studies have also examined cytosine methylation in Sulfolobus acidocaldarius (Grogan, 2003 ), the structure of a type I S subunit in Methanococcus jannaschii (Kim et al, 2005 ), and the activity of a type II methyltransferase in a virus infecting Natrialba magadii (Baranyi et al, 2000 ).…”