Single-stranded DNA binding and methylation by EcoP1I DNA methyltransferase

Sistla, Srivani; Krishnamurthy, Vinita; Rao, Desirazu N.

doi:10.1016/j.bbrc.2003.12.070

Cited by 7 publications

(5 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The enzyme showed maximal activity in the presence of Mg 2+ and Ca 2+ whereas in the presence of Mn 2+ , M.EcoP1I methylated DNA less efficiently. It has been shown that M.EcoP1I binds to DNA in the presence of various metal ions with varying affinities [6]. Our results and earlier studies suggest that M.EcoP1 harbors a metal binding center which is important for methylation.…”

Section: Resultssupporting

confidence: 68%

“…The 1948 bp long M.EcoP1I gene was amplified by a polymerase chain reaction (PCR) using pVK1 construct [6] containing a ~2 kb long EcoP1I mod gene as a template with Phusion DNA polymerase, using the forward primer and reverse primer (Table 1). The primers were designed with the help of the annotated complete sequence of the P1 prophage by identifying the putative gene sequence of M.EcoP1I .…”

Section: Methodsmentioning

confidence: 99%

“…One of the interesting features of Type III R-M systems is that they recognize asymmetric recognition sequences and methylate only one strand [5]. Interestingly, M.EcoP1I binds to both double- and single-strand DNA substrates containing the asymmetric recognition sequence 5′-AGACC-3′ and methylates them with almost similar efficiency [6]. Based on the linear arrangement of conserved motifs, M.EcoP1I belongs to the β -subgroup of exocyclic amino MTases (catalytic motif-target recognition domain-AdoMet binding motif).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Kinetics of Methylation by EcoP1I DNA Methyltransferase

et al. 2010

Self Cite

View full text Add to dashboard Cite

EcoP1I DNA MTase (M.EcoP1I), an N6-adenine MTase from bacteriophage P1, is a part of the EcoP1I restriction-modification (R-M) system which belongs to the Type III R-M system. It recognizes the sequence 5′-AGACC-3′ and methylates the internal adenine. M.EcoP1I requires Mg2+ for the transfer of methyl groups to DNA. M.EcoP1I is shown to exist as dimer in solution, and even at high salt concentrations (0.5 M) the dimeric M.EcoP1I does not dissociate into monomers suggesting a strong interaction between the monomer subunits. Preincubation and isotope partitioning studies with M.EcoP1I indicate a kinetic mechanism where the duplex DNA binds first followed by AdoMet. Interestingly, M.EcoP1I methylates DNA substrates in the presence of Mn2+ and Ca2+ other than Mg2+ with varying affinities. Amino acid analysis and methylation assays in the presence of metal ions suggest that M.EcoP1I has indeed two metal ion-binding sites [358ID(x)n … ExK401 and 600DxDxD604 motif]. EcoP1I DNA MTase catalyzes the transfer of methyl groups using a distributive mode of methylation on DNA containing more than one recognition site. A chemical modification of EcoP1I DNA MTase using N-ethylmaleimide resulted in an irreversible inactivation of enzyme activity suggesting the possible role of cysteine residues in catalysis.

show abstract

Section: Resultssupporting

confidence: 68%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Kinetics of Methylation by EcoP1I DNA Methyltransferase

et al. 2010

Self Cite

View full text Add to dashboard Cite

show abstract

“…Our surprising demonstration that CcrM binds single-stranded DNA (ssDNA) with tight affinity and specificity (Figure A,D and Table A,B,D,E) led to an effort to challenge this provocative finding. The ability of DNA MTases and other enzymes to modify ssDNA and dsDNA is not novel, although it is rarely reported, particularly for enzymes involving sequence-specific recognition. ,− Unfortunately, with few exceptions, the basis for claiming such dual specificities often relies on the use of large, biological DNA (e.g., viral genomes and plasmids), which can take on transient duplex structures. For CcrM, the K m ssDNA (17.22 ± 2.4 nM) and k cat (apparent, 3.9 ± 0.2 min –1 ) are similar to the values for dsDNA (Figure B), although in combination with increases in k methylation , the enzyme generally shows slightly smaller specificity constants ( k methylation / K d DNA ) for ssDNA than for the identical dsDNA sequence (Table B,C).…”

Section: Discussionmentioning

confidence: 99%

Caulobacter crescentus Cell Cycle-Regulated DNA Methyltransferase Uses a Novel Mechanism for Substrate Recognition

2017

View full text Add to dashboard Cite

Caulobacter crescentus relies on DNA methylation by the cell cycle-regulated methyltransferase (CcrM) in addition to key transcription factors to control the cell cycle and direct cellular differentiation. CcrM is shown here to efficiently methylate its cognate recognition site 5'-GANTC-3' in single-stranded and hemimethylated double-stranded DNA. We report the K, k, k, and K for single-stranded and hemimethylated substrates, revealing discrimination of 10-fold for noncognate sequences. The enzyme also shows a similar discrimination against single-stranded RNA. Two independent assays clearly show that CcrM is highly processive with single-stranded and hemimethylated DNA. Collectively, the data provide evidence that CcrM and other DNA-modifying enzymes may use a new mechanism to recognize DNA in a key epigenetic process.

show abstract

“…Moreover, the endonuclease and methyltransferase sub-units are part of the same complex, with the Mod subunit providing specificity. Type III EcoPI was shown to methylate ssDNA in vitro (67). Thus, the dynamics of restriction and methylation on incoming plasmids might make conjugative entry intrinsically less susceptible to Type III restriction, similarly to filamentous, ssDNA phages which are also relatively resistant to Type III restriction (68).…”

Section: Discussionmentioning

confidence: 99%

Various plasmid strategies limit the effect of bacterial Restriction-Modification systems against conjugation

Dimitriu,

Szczelkun,

Westra

2024

Preprint

View full text Add to dashboard Cite

In bacteria, genes conferring antibiotic resistance are mostly carried on conjugative plasmids, mobile genetic elements which spread horizontally between bacterial hosts. Bacteria carry defence systems which defend them against genetic parasites, but how effective these are against plasmid conjugation is poorly understood. Here, we study to what extent Restriction-Modification (RM) systems – by far the most prevalent bacterial defence systems - act as a barrier against plasmids. Using 10 different RM systems and 13 natural plasmids conferring antibiotic resistance inEscherichia coli, we uncovered variation in defence efficiency ranging from none to 105-fold protection. Further analysis revealed genetic features of plasmids that explain the observed variation in defence levels. First, the number of RM recognition sites present on the plasmids generally correlates with defence levels, with higher numbers of sites being associated with stronger defence. Secondly, some plasmids encode methylases that protect against restriction activity. Finally, we show that a high number of plasmids in our collection encode anti-restriction genes that provide protection against several types of RM systems. Overall, our results show that it is common for plasmids to encode anti-RM strategies, and that, as a consequence, RM systems form only a weak barrier for plasmid transfer by conjugation.

show abstract

Single-stranded DNA binding and methylation by EcoP1I DNA methyltransferase

Cited by 7 publications

References 31 publications

Kinetics of Methylation by EcoP1I DNA Methyltransferase

Kinetics of Methylation by EcoP1I DNA Methyltransferase

Caulobacter crescentus Cell Cycle-Regulated DNA Methyltransferase Uses a Novel Mechanism for Substrate Recognition

Various plasmid strategies limit the effect of bacterial Restriction-Modification systems against conjugation

Contact Info

Product

Resources

About