Multispecific DNA methyltransferases from Bacillus subtilis phages

Abstract

Temperate Bacillus subtilis phages SPR, q3T, e l l and SPB code for DNA methyltransferases, each having multiple sequence specificities. The SPR wild-type and various mutant methyltransferases were overproduced 1 000-fold in Escherichia coli and were purified by three consecutive chromatographic steps.The stable form of these multispecific enzymes in solution are monomers with a relative molecular mass (M,) of about 50000. The methyl-transfer kinetics of the SPR wild-type and mutant enzymes were determined with DNA substrates carrying either none or one of the three recognition sequences (GGCC, CCGG, CC,^CG).Evaluation of the catalytic properties for DNA and S-adenosylmethionine binding suggested that the N H1-terminal part of the protein is important for both non-sequence-specific DNA binding and S-adenosylmethonine binding as well as transfer of methyl groups. On the other hand, mutations in the COOH-terminal part lead to weaker site-specific interactions of the enzyme.Antibodies raised against the purified SPR enzyme specifically immunoprecipitated the q3T, el 1 and SPB methyltransferases, but failed to precipitate the chromosomally coded enzymes from B. subtilis (BsuRI) and B. sphaericus (BspRI). Immunoaffinity chromatography is an efficient purification step for the related phage methyltransferases.The temperate Bacillus subtilis phages SPR, q3T, el 1 and SPP have evolved a protection mechanism against host restriction enzymes by self-methylating their DNA at various sequences [l]. q3T, e l l and SPP methyltransferases have the potential to methylate the sequences GGCC (HaeIIIIBsuRI) and GCNGC (Fnu4HI) [2], while the SPR methyltransferase recognizes three sequences: GGCC [3], CCGG (HpaII/ MspIIBsuF) [4, 51 and C&GG (EcoRII) (U. Gunthert and L. Reiners, unpublished results). The asterisk denotes the position of methylation.Like the related type I1 modification enzymes, the phagederived methyltransferases are composed of a single polypeptide. As calculated from the DNA sequence, the SPR methyltransferase gene encodes a 439-residue protein with a M , = 49826 [6]. An approximate M , of 47000 for the q3T, e l l and SPP enzymes has been determined by minicell expression studies and in an in vitro transcription/translation system [7].A set of SPR methyltransferase mutants has been isolated and the positions of the mutations localized in the methyltransferase coding region [6] represented in this study by the SPR26 methyltransferase [3], is affected in its ability to methylate all three sequences. Another group of mutants is altered in their ability to interact with either the GGCC sequence (SPR19) [4] or the CCGG sequence (SPR83) [6]. A mutation which affects methylation at CC&G has not yet been identified. Another mutation (a frame-shift) has been isolated, which codes for a truncated protein of M, 28000 (SPRlOl) [6] showing reduced sitespecific methylation (U. Gunthert and L. Keiners, unpublished results).The goal of our research is the understanding of interactions between enzymes and DNA and these methyltrans€...

Identification of a subdomain within DNA-(cytosine-C5)-methyltransferases responsible for the recognition of the 5′ part of their DNA target.

“…In order to activate the chimeric enzyme, we introduced amino acid exchanges, insertions and deletions into TRD EF, to assimilate its sequence stepwise to the corresponding sequence of TRD E (Figure 4). For an analysis of the DNA methylation capacities of the resulting mutant derivatives of M.SPR-EF (M.SPR-EF-1 to -EF-8) we tested the in vitro methylation activity of Escherichia coli cells overexpressing the different mutant proteins as described previously (Gunthert et al, 1986). These analyses revealed that alterations of amino acids within regions I and II or in both regions (M.SPR-EF-1-4) had no activating effect.…”

A genetic and functional analysis of the unusually large variable region in the M·Alu I DNA-(cytosine C5 )-methyltransferase

“…The Mcr A Dcm A E. coli strain GM2929 (Palmer and Marinus 1994) was used to check the phenotype of hybrids containing TRD E. Plasmid pKB131 (Gunthert et al 1981(Gunthert et al , 1986aBuhk et al 1984;Posfai et al 1984) kindly provided by Dr. T. Trautner, carries the gene for the SPR DNA methylase and is a derivative of pBR328. Plasmid pTrc99-SPRMT was generated by moving the sprM gene into pTrc99A (Gunthert et al 1986b;Balganesh et al 1987;Trautner et al 1988a, b;Wilke et al 1988).…”

“…In both 5mC and amino methylases, the largest variable region lies immediately downstream of conserved motif VIII and contains one or more Target Recognizing Domains (TRDs) responsible for DNA sequence speci®city (Gunthert et al 1986a;Wilke et al 1988;Lauster et al 1989;Klimasauskas et al 1991;Mi and Roberts 1992). When individual TRDs were identi®ed and their boundaries de®ned by determining the eects of mutations and chimeric constructs, multispeci®c methylases were found to have as many as ®ve active TRDs Trautner et al 1988a, b;Wilke et al 1988), while monospeci®c methylases appeared to have just one (Klimasauskas et al 1991;Kumar et al 1992;Mi and Roberts 1992).…”