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A protein architecture guided screen for modification dependent restriction endonucleases.

Thomas Lutz1, Kiersten Flodman1, Alyssa Copelas1

  • 1New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.

Nucleic Acids Research
|September 11, 2019
PubMed
Summary
This summary is machine-generated.

Researchers identified novel fusion proteins with DNA restriction capabilities. Some enzymes target modified DNA bases like 5-methylcytosine, while others, despite lacking endonuclease activity, confer phage resistance.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Modification dependent restriction endonucleases (MDREs) often possess distinct catalytic and modification-sensing domains.
  • Uncharacterized fusion proteins with potential restriction-modification activities require systematic investigation.

Purpose of the Study:

  • To identify and characterize novel fusion proteins with PUA or DUF3427 domains and HNH or PD-(D/E)XK catalytic domains.
  • To elucidate the DNA modification-dependent activities and biological roles of these novel enzymes.

Main Methods:

  • Systematic search for fusion proteins combining specific domain families (PUA/DUF3427 and HNH/PD-(D/E)XK).
  • Clustering of enzymes based on modification-sensing domain similarity.
  • Assays to determine DNA cleavage activity and phage restriction capabilities.

Main Results:

  • PUA-containing enzymes (TspA15I, ScoA3IV, YenY4I groups) cleaved DNA with 5-methylcytosine or 5-hydroxymethylcytosine.
  • A distinct PUA-containing enzyme (ScoA3V) showed nicking activity stimulated by 6-methyladenine.
  • DUF3427-containing proteins exhibited minimal endonuclease activity but conferred potent phage resistance, often located in defense islands.

Conclusions:

  • Fusion proteins with PUA domains function as modification-dependent endonucleases targeting specific DNA base modifications.
  • DUF3427-containing proteins, despite lacking significant endonuclease activity, play a role in bacterial defense mechanisms.
  • The genomic context of these fusion proteins supports their roles in restriction-modification systems and phage defense.