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Immunostaining for DNA Modifications: Computational Analysis of Confocal Images
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Plasmid replication-associated single-strand-specific methyltransferases.

Alexey Fomenkov1, Zhiyi Sun1, Iain A Murray1

  • 1New England Biolabs Inc., 240 County Road, Ipswich, MA, USA.

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|December 3, 2020
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Summary
This summary is machine-generated.

Two unusual DNA methyltransferases (MTases) modify single DNA strands asymmetrically, with activity dependent on replication origins. This discovery sheds light on DNA modification and plasmid establishment in bacteria.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Microbiology

Background:

  • Pathogenic bacteria like Burkholderia cenocepacia and Escherichia coli harbor unique genes on plasmids.
  • Unusual DNA methyltransferase (MTase) genes, M.BceJIII and M.EcoGIX, were identified on plasmids pBCA072 and pESBL, respectively.

Purpose of the Study:

  • To investigate the enzymatic activity and in vivo behavior of M.BceJIII and M.EcoGIX.
  • To understand the factors influencing the asymmetric DNA methylation patterns observed.
  • To explore the role of M.EcoGIX in plasmid establishment and host interaction.

Main Methods:

  • Pacific Biosciences Single-Molecule Real-Time (SMRT) sequencing was employed to analyze DNA methyltransferases.
  • Artificial constructs were used to study enzyme activity.
  • Genetic and biochemical assays were performed to assess enzyme function and interactions.
  • Mating experiments with engineered plasmid derivatives were conducted.

Main Results:

  • Both M.BceJIII and M.EcoGIX exhibit promiscuous N6-methyladenine (m6A) modification of single DNA strands in the SAY motif.
  • In vivo, methylation is asymmetric, occurring on only one DNA strand and is incomplete (~40% modification).
  • Enzyme activity is dependent on plasmid replication mode; DNA Polymerase I (PolI)-dependent origins support asymmetric modification, while PolI-independent origins do not.
  • M.EcoGIX facilitates plasmid pESBL establishment by blocking restriction enzymes in an orientation-dependent manner.

Conclusions:

  • A potential MTase-PolI complex may distinguish between different plasmid replication origins.
  • Asymmetric single-strand DNA methylation by these MTases is influenced by replication machinery.
  • M.EcoGIX plays a role in conjugal transfer and plasmid establishment by interacting with the host DNA replication and restriction systems.