Agmenellum quadruplicatum M.AquI, a novel modification methylase

Abstract

The complete type II modification methylase of Agmenellum quadruplicatum was cloned in Escherichia coli as an R Sau3A fragment of approximately 4.5 kilobases. The coding sequence was contained in a stretch of 1,156 base pairs which was organized into two parallel, partly overlapping open reading frames of 248 and 139 codons. In vivo complementation experiments showed that the synthesis of both predicted peptides was required for full methylase activity. The amino acid sequences were considerably similar to regions of other deoxycytidylate methylases.In procaryotic cells, two fundamentally different restriction-modification (R-M) systems have been found. They can be distinguished by their need for cofactors, as well as by their genetic organization. The R-M complex of the so-called type I systems requires S-adenosylmethionine, ATP, and Mg2+ for its functioning and contains the products of three genes: the first codes for the S unit, which recognizes the specific site, the second directs the synthesis of the R unit, which is responsible for cutting unmethylated DNA, and the last is translated into the M unit, which methylates non-or hemimethylated DNA. The absence of both of the protecting methyl groups at the recognition sites primes the R-M complex for attacking the DNA; the actual cleavage, however, does not occur at the recognition site itself, but somewhere else in the DNA molecule. The type II systems, on the other hand, code for two proteins: an endonuclease needing only Mg2+ for cleavage (which takes place at the recognition site itself) and a modification methylase requiring only S-adenosylmethionine for the protection of DNA. All type II systems characterized at the genetic level so far have been found to be encoded by just two genes: one for the endonuclease and another for the methylase. Only in the case of DpnII have two different methylase genes been discovered (3); both code for a protein that methylates independently of the other.In the cyanobacterium Agmenellum quadruplicatum, an R-M system that consists of isoschizomers of the AvaI (10,13)

Recombinant α and β Subunits of M.AquI Constitute an Active DNA Methyltransferase

“…In our experiments, we used both subunits in a purified form. Our in vitro MTase assay results support Karreman and De Waard's (1990) conclusions. As seen in Table 1, a proportion of radioactivity that was being eluted from the gel filtration column in the first two fractions shows that M.AquI incorporated 3 H methyl groups into calf thymus DNA when both the α and the β subunits were present in the reaction mixture.…”

“…1). Karreman and De Waard (1990) reported the molecular cloning and sequence analysis of M.AquI. Their result indicated that this enzyme comprises two polypeptides, termed α and β.…”

“…Although a typical C5-DNA MTase is encoded by a single ORF, another bacterial MTase M.EcoHK311 has also been reported to require two polypeptides for in vitro methylation of target DNA (Lee et al, 1995(Lee et al, & 1996. Karreman and De Waard (1990) demonstrated that M.AquI is comprised of two polypeptide chains, which constitute an active MTase. However, purified subunits were not used in their experiments.…”

“…Since the monospecific DNA MTase M.AquI is a heterodimer; therefore, in principle, this enzyme affords the opportunity to dissect out the determinants of DNA recognition among the monospecific DNA MTases. A comparison between the predicted amino acid sequences of the α and β subunits of M.AquI (Karreman and De Waard, 1990) and the sequence of the related enzyme M.HhaI (Cheng et al, 1993), revealed that the α subunit constituted most of the AdoMet binding site, and contained the catalytic cysteine. The β polypeptide contained the TRD sequence together with homology blocks IX and X, including the C-terminal helixloop-helix region.…”

“…Tel: +90-3462191010(1077); Fax: E-mail: hpinar@cumhuriyet.edu.tr methionine (AdoMet) as a methyl donor, and the outermost cytosine residue in the CYCGRG sequence is the acceptor for the methyl group. The M.AquI gene was isolated as a 1.2 kb DNA fragment, and the complete nucleotide sequence was determined (Karreman and De Waard, 1990). It is encoded by two partly overlapping ORFs (termed α and β).…”