M1.MboII and M2.MboII type IIS methyltransferases: different specificities, the same target

Abstract

Methylation of a base in a specific DNA sequence protects the DNA from nucleolytic cleavage by restriction enzymes recognizing the same sequence. The MboII restriction-modification (R-M) system of Moraxella bovis ATCC 10900 consists of a restriction endonuclease gene and two methyltransferase genes. The enzymes encoded by this system recognize an asymmetrical sequence 59-GAAGA-39/39-CTTCT-59. M1.MboII modifies the last adenine in the recognition sequence 59-GAAGA-39 to N 6 -methyladenine. A second methylase, M2.MboII, was cloned and purified to electrophoretic homogeneity using a four-step chromatographic procedure. It was demonstrated that M2.MboII modifies the internal cytosine in the recognition sequence 39-CTTCT-59, yielding N 4 -methylcytosine, and moreover is able to methylate single-stranded DNA. ) inhibit M2.MboII methylation activity. It was found that the isomethylomer M2.NcuI from Neisseria cuniculi ATCC 14688 behaves in the same manner. Functional analysis showed that the complete MboII R-M system, consisting of two methyltransferases genes and the mboIIR gene, is the most stable and the least harmful to bacterial cells.

Identification of Restriction-Modification Systems of Bifidobacterium animalis subsp. lactis CNCM I-2494 by SMRT Sequencing and Associated Methylome Analysis

“…lactis CNCM I-2494 just one methyltransferase-encoding gene was present in the BanLI R-M system, and it may just be that the second methylase-encoding gene was lost due to an integration event upstream of banLI.M . It is thought that recognition and methylation of strand-specific sequences by type IIS R-M systems is advantageous to bacterial cells, as strands generated during DNA replication can be simultaneously protected from attack by site-specific endonucleases [54]. The prototype type IIS R-M systems is FokI, while others, such as the HgaI, MboII LlaI and LlaJI type IIS R-M systems, have been well characterized.…”

“…The prototype type IIS R-M systems is FokI, while others, such as the HgaI, MboII LlaI and LlaJI type IIS R-M systems, have been well characterized. [54]–[57]. The FokI R-M system recognizes an asymmetrical sequence and cleaves the DNA at a specific distance from the recognition sequence.…”

“…In this instance the protein sequences of the two methylases are conserved suggesting that these have evolved as a result of gene duplication [59]. Similarly, MboII, identified from Moraxella bovis ATCC 10900 recognizes an asymmetrical sequence, and this system comprises of two methyltransferase encoding genes m1.mboII and m2.mboII , the former specifying an adenine methyltransferase while m2.mboII encodes a cytosyl-methyltransferase [54]. Type IIS R-M systems have also been found to be encoded on conjugative plasmids in Lactococcus lactis , the LlaI R-M system was identified on a 46.2 kb plasmid, pTR2030 [60], [61], while the LlaJI R-M system was identified as one of three phage resistance systems present on the 65 kb plasmid, pNP40 [55]–[57].…”

RNA editing by T7 RNA polymerase bypasses InDel mutations causing unexpected phenotypic changes

“…Here, we describe another aspect of T7 RNAP that was revealed after mboIIM2 DNA methyltransferase of Moraxella bovis expression analyses ( 29 , 30 ). In light of the results presented here, bacteria with a target gene carrying a single or double insertion/deletion (InDel) mutation that should cause frameshift wild type reading frame are phenotypically heterogenous.…”

Kinetics of Methylation by EcoP1I DNA Methyltransferase

“…The presence of metal ions is not an absolute requirement for M.BcgI [29], M.AloI [30], M.Eco57I [31], and M.MmeI [32] but the methylation activity of these enzymes was enhanced in the presence of Mg 2+ . On the other hand, M.HindIII, M.LlaCI, M.EcoVIII [33], M.PabI [18], M1.MboI, M2.MboII, M1.NcuI, M.NcuII [34, 35], and M.FokI [36] showed inhibition of methylation activity in the presence of various metal ions. It is clear that the activities of DNA MTases characterized so far respond differently in the presence of metal ions.…”