Since the annotation of the Mu genome in 2002 (17
), Mu-like transposable phage family members have been identified during the sequencing of several bacterial genomes. Comparison of these Mu-like phages, prophages and phage-related elements indicates that their genome organizations are similar.
The 1332-bp fragment amplified from genomic DNA of the H. influenzae
ATCC 11116 strain analyzed in this study was highly similar (86% identity) to a segment of the H. influenzae
F3031 genomic island containing genes related to phage Mu (18
). This high level of sequence similarity indicated that this ATCC 11116 genomic fragment represents part of a complete or defective Mu-like prophage present in this bacterial host.
Mu-like prophages often carry genes homologous to those of phage Mu and also other apparently unrelated genes that appear to perform analogous functions (17
). Two of ORFs (nma1821
) characterized in this study are present at the right end of two Mu-like prophage genomes (17
), and the third (hia5
) is predicted to be a part of a Mu-like sequence. All three localize in the prophage genomes at positions syntenic to the mom
gene of Mu. The putative protein products of the hia5
genes also share sequence similarity, but are not homologous to the Mom protein of Mu—the modifying enzyme that converts adenine residues in DNA to acetamidoadenine. In this study, we cloned, expressed and characterized the hia5
genes and identified their products as novel DNA MTases with extremely relaxed specificity.
According to the nature of the reactions they catalyze, DNA MTases can be divided into three separate groups, generating m6
C or m5
C. All known DNA MTases have conserved motifs, of which I–VIII and X are common to most subfamilies (33
). X-ray crystallographic analyses have revealed that the region containing the conserved motifs corresponds to the structurally and evolutionarily conserved catalytic domain, in which motifs X and I–III are involved in AdoMet binding and motifs IV–VIII form the active site. The common AdoMet binding region is conserved across the whole MTase superfamily, while the ‘catalytic motifs’, especially motif IV, exhibit subfamily-specific patterns (33
The enzymes that were the subject of this study belong to a previously uncharacterized subfamily of DNA adenine MTases. Despite their relatively distant sequence similarity to characterized MTases, they possess the characteristic signature motifs, including the catalytic motif IV (DPPY), which is typical for MTases that modify amino groups in various substrates, including RNAs and proteins. We have shown that in vitro these enzymes catalyzed the transfer of methyl groups from an AdoMet donor to the substrate DNA, and that they modified only adenine residues, converting them into m6A. To further confirm that the Hia5, Hin1523 and Nme1821 proteins act as adenine MTases, we altered their predicted catalytic sites by site-directed mutagenesis. Substitution of aspartate in the DPPY motifs with alanine (point substitution D194A for Hia5 and Hin1523, and D191A for Nme1821) completely abolished the adenine MTase activity of these enzymes.
To define the target sequences of these novel MTases, we used an endonuclease protection assay. All 30 REases with previously established sensitivity to m6A in their recognition sequences were unable to cleave DNAs isolated from strains expressing the tested genes or only a partial cleavage was observed. On the contrary, the control REases, which are insensitive to m6A, were able to cleave the DNAs to produce the expected restriction patterns. These results were the first indication of the massive DNA methylation catalyzed by the Hia5, Hin1523 and Nme1821 enzymes. Corroborating evidence was obtained using an HPLC DNA methylation assay to evaluate the base composition of λ DNA methylated with Hia5 in vitro. This analysis revealed that as much as 61% of the adenine residues were converted to m6A (29.6% by Hin1523). In other words more than every second adenine in λ DNA was modified by the Hia5 enzyme, which strongly suggested that a predicted sequence specificity cannot be longer than a dinucleotide. To directly test this assumption, we used substrates with repetitions of the dinucleotides CA, GA, TA and AA (poly-A tract). The first two were methylated by the Hia5 enzyme with comparable rates and TA was methylated slightly slower. This slight difference could be associated with the nature of the substrate. Adenines are present in both the upper and lower strands of the TA50 duplex, unlike in the CA10 and GA10 duplexes, where they are present only in one strand. It is possible that only one of the two diagonally arranged adenines in the TA duplex is methylated efficiently, while the other may be methylated at a lower rate.
Hin1523 also methylated the aforementioned duplexes, although the rate of CA10 methylation was the fastest. The TA50 duplex was modified by Hin1523 slightly slower similar to the Hia5 enzyme. Weak discrimination against the GA10 duplex observed for the Hin1523 does not exclude GA dinucleotides as substrates, since the GA10 duplex methylation rate was only two times lower than the rates measured for the CA10 and TA50 duplexes.
The incorporation signal obtained for the AA21 substrate (poly(A)/poly(T) tract) was at the background level. Adenines occurring in the Cm2A1 and Cm2A3 duplexes also appeared to be poor substrates for both the Hia5 and Hin1523 enzymes, suggesting that internal adenines within poly(A)-stretches are effectively not methylated.
Our results suggest that all adenine residues in double-stranded DNA, with the exception of poly(A)-tracts, constitute potential substrates for the Hia5 and Hin1523 enzymes, and that these enzymes are indeed sequence non-specific. Formally, their potential ‘sequence specificity’ could be summarized as AB or BA (where B
C, G or T). 71% of adenines in λ DNA occur in AB (BA) sequences. Sixty-one percent level of Hia5 λ DNA methylation in HPLC assay is close to this value.
To our knowledge, this is the first example of an exocyclic amino MTase with such extraordinary sequence promiscuity. High levels of adenine residue methylation in vivo
and in vitro
in a heterologous host (E. coli
) have been described for M.CviQII that modifies the dinucleotide sequence A
), but this was demonstrated only by a restriction protection assay and the extent of M.CviQII modification has never been quantified.
REases are commonly used as molecular biology tools and their sensitivity to methylation has to be considered when digesting DNA, because cleavage may be blocked or impaired when a particular base in the recognition site is methylated. Our results suggest that the novel MTases characterized in this study can be very useful in verifying the adenine methylation sensitivity of many REases and in revealing such behavior in those endonucleases with unknown sensitivity to this DNA modification. Indeed, we demonstrated that 16 REases with unknown sensitivity to m6A in their cognate sequences were in fact sensitive to this modification. The knowledge whether a particular REase is sensitive to a nucleotide modification in its recognition sequence may be utilized to analyze patterns of regional methylation or to establish the methylation status of genomic DNA.
In eukaryotic cells, nuclear DNA is subject to enzymatic methylation resulting in the formation of m5
C residues, mainly in CG and CNG sequences. In plants and animals, this DNA methylation is species-, tissue- and even organelle-specific. It changes with age and is regulated by hormones. On the other hand, genome methylation can control hormonal signaling (36
It has long been known that m6
A is present in the DNA of algae and their viruses, fungi and protists. Furthermore, and in spite of the common opinion that m6
A is not found in the DNA of higher eukaryotes, this modified base has been detected in plastid, mitochondrial and nuclear plant DNA and in mosquito DNA (37
). Accumulating evidence suggests that m6
A affects the regulation of gene expression in mammalian cell (38–40
). The use of the novel MTases identified in this work might allow us to study the effects of methylation on eukaryotic DNA–protein interactions and, in consequence, its impact on transcription in mammalian or plant cells.
Sequence searches with the Hia5 sequence against a non-redundant database (NCBI) revealed many closely related proteins, suggesting that there may exist more DNA
A MTases with similar properties, i.e. extremely relaxed substrate specificity. All identified Hia5 homologs were present in bacterial parasites, pathogens or commensal strains in mammals, including humans (data not shown). Thus, the contribution of Mu-like sequences to the virulence of their bacterial hosts should be considered.
Despite hosting Mu-like prophages encoding these novel MTases, we did not observe additional methylation of DNA during the normal growth of H. influenzae
Rd (lysogenic for FluMu) and H. influenzae
biotype aegyptius ATCC 11116, or after induction with mitomycin C, beyond the native methylation resulting from the activities of M.HinDam and M.HindIII, and M.HaeII, respectively. We were also unable to isolate FluMu phage particles (data not shown). Similarly, McGillivary et al.
) failed to purify phage particles from the H. influenzae
biotype aegyptius F3031. The expression of the mom
gene is under strict control mediated by the Com protein (11
). Possible Com (or Com homolog) binding sites and associated regulatory elements have been identified at the 5′-end of the hin1523
). This suggests that the Mom protein of Mu and these novel MTases could be regulated by a similar mechanism and may explain why we were unable to detect in vivo
MTase activity under laboratory conditions; the hin1523
gene presumably remains repressed until the prophage enters the lytic cycle.
Saariaho et al.
) revealed that FluMu encodes a functional transposase and contains critical transposase binding sites, but they were also unable to obtain FluMu virus plaques. Other attempts to induce Mu-like prophage excision and cell lysis using the DNA-damaging agents mitomycin C and UV irradiation have also been unsuccessful (42
). Under certain conditions it is likely that the host may benefit from the presence of prophages (in particular those unable to enter a lytic cycle) by conferring immunity to superinfection by related phages (43
Although the biological role of these newly characterized MTases for Mu-like phages and their hosts remains unknown, the discovery of a group of DNA:m6A MTases with extremely relaxed substrate specificity has significance due to their potential as tools in molecular biology research, particularly in the study of the poorly characterized role of adenine methylation in eukaryotic DNA.