Dual nucleotide specificity determinants of an infection aborting anticodon nuclease

Ekaterina Krutkina, Daniel Klaiman, Tamar Margalit, Moran Jerabeck-Willemsen, Gabriel Kaufmann

Research output: Contribution to journalArticlepeer-review

Abstract

The anticodon nuclease (ACNase) PrrC is silenced by a DNA restriction-modification (RM) protein and activated by a phage T4-encoded restriction inhibitor. The activation is driven by GTP hydrolysis while dTTP, which accumulates during the infection, stabilizes the active form. We show here, first, that the ABC-ATPase N-domains of PrrC can accommodate the two nucleotides simultaneously. Second, mutating a sequence motif that distinguishes the N-domain of PrrC from typical ABC-ATPases implicates three residues in the specificity for dTTP. Third, failure to bind dTTP or its deprivation hastened the centrifugal sedimentation of PrrC, possibly due to exposed sticky PrrC surfaces. Fourth, dTTP inhibited the GTPase activity of PrrC, probably by preventing GDP from leaving. These observations, correlated with relevant traits of a related ACNase, further suggest that PrrC utilizes GTP at canonical ABC-ATPase sites and binds dTTP to distinct sites exposed upon disruption of the ACNase-silencing interaction with the RM partner.

Original languageEnglish
Pages (from-to)260-272
Number of pages13
JournalVirology
Volume487
DOIs
StatePublished - 1 Jan 2016

Keywords

  • ABC-ATPase
  • Anticodon nuclease
  • DNA restriction and anti-restriction
  • DTTP binding
  • Phage T4-exclusion
  • PrrC

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