New ATP-dependent, multi-subunit restriction-modification systems that use split DNA methyltransferases for host protection

In prokaryotes, host-dependent restriction-modification (HDRM) systems comprise a major category of defense against DNA invasion. Such systems monitor the pattern of DNA modification conferred by site-specific DNA methyltransferases (MTase), attacking with nuclease when specific sites do not carry the host modification pattern. NTPase activity is often used to regulate activation of nuclease activity. Bioinformatic analysis yielded provisional identifications of nuclease, MTase and NTPase domains in a new configuration of component domains with novel connectivity. Confirmation of domain activity employed in vitro transcription-translation of epitope-tagged proteins yielding assignment of catalytic activity to protein segments. In vivo heterologous expression of the corresponding protein segments followed by affinity purification allowed us to characterize DNA site-requirements for cleavage, ATPase and MTase activities. Three representatives from different phyla employ methylating MTase components divided into two parts, with the parts connected to regulatory NTPase and defensive nuclease domains in novel organizations. The DrdVI system from Deinococcus wulumuqiensis NEB479 (formerly Deinococcus radiodurans ) recognizes 5’-GCAGCC-3’, modifying only one strand. Two systems, PenI ( Porphyromonas endodontalis ATCC 35406) and SmoLII ( Streptobacillus moniliformis DSM12112), each recognize 5’-GCAGT-3’, again modifying the A of that strand. Organizationally, the genes and proteins of these MTase activities have separated catalytic motifs from DNA-binding components in distinct ways. All have a component subunit that connects an endonuclease to an NTP-hydrolyzing translocase domain. The endonucleases of DrdVI and PenI require (d)ATP and divalent cations (Mg 2+ or Mn 2+ ) as cofactors. Cleavage occurs when head-to-head inverted recognition sites are present. DNA cleavage does not occur at a fixed site. The one-strand modification and inverted cleavage site configuration resemble typical Type III enzymes, but the connectivity of domain components sets them apart. Bioinformatic analysis reveals numerous similar putative HDRM systems. This suggests that they may represent a new subtype containing features of both Type I and Type III systems.