The acidic C-terminus of vaccinia virus I3 single-strand binding protein promotes proper assembly of DNA–protein complexes

Abstract

The vaccinia virus I3L gene encodes a single-stranded DNA binding protein (SSB) that is essential for virus DNA replication and is conserved in all Chordopoxviruses. The I3 protein contains a negatively charged C-terminal tail that is a common feature of SSBs. Such acidic tails are critical for SSB-dependent replication, recombination and repair. We cloned and purified variants of the I3 protein, along with a homolog from molluscum contagiosum virus, and tested how the acidic tail affected DNA-protein interactions. Deleting the C terminus of I3 enhanced the affinity for single-stranded DNA cellulose and gel shift analyses showed that it also altered the migration of I3-DNA complexes in agarose gels. Microinjecting an antibody against I3 into vaccinia-infected cells also selectively inhibited virus replication. We suggest that this domain promotes cooperative binding of I3 to DNA in a way that would maintain an open DNA configuration around a replication site.

The Vaccinia virion: Filling the gap between atomic and ultrastructure

“…Vaccinia DNA ligase (DNLI), a nick sealing protein, showed 22 crosslinking partners (S1 Fig)—an unexpectedly large number for a specialized enzyme. Among these were four other members of the DNA binding/DNA metabolism group (Table 3), namely K4—the Vaccinia DNA nicking enzyme for genome telomeres [109], I1 –a Vaccinia telomere binding protein, I3 –an ssDNA binding protein [110, 111] and Vaccinia topoisomerase TOP1. Moreover, direct crosslinking was detected between I1 and I6, both of which are known to bind Vaccinia genome telomeres ([2, 109] and references therein).…”

Type IV Coupling Proteins as Potential Targets to Control the Dissemination of Antibiotic Resistance

“…The CTDs of different conjugative systems can present different functions. Many DNA-binding proteins show acidic C-terminal sequences that take part in the DNA-binding process (Lee and Thomas, 2000;Wang et al, 2007;Harrison et al, 2016;Basu et al, 2020), which appears to be their main function in T4CPs. For example, in the case of TraD F T4CP, the interaction of its CTD with one of the components of the relaxosome, TraM F , is essential for recognition and plasmid transfer (Lu and Frost, 2005;Lu et al, 2008).…”

Isolation and Characterization of vΔI3 Confirm that Vaccinia Virus SSB Plays an Essential Role in Viral Replication

“…In many systems, SSBs have charged C=-terminal tails that mediate protein-protein interactions. In the case of I3, no such interactions have been found, and instead the C=-terminal tail has been shown to modulate DNA binding, as has also been shown for some prokaryotic SSBs (16). Another area of study for the future is to assess the repertoire of proteins that I3 might interact with during viral replication.…”

“…The viral A50 protein is a DNA ligase, and it too is essential for viral DNA replication in those circumstances in which the cellular DNA ligase I is not available; an enzymatically active DNA ligase is needed for productive infection (12;Czarnecki and Traktman,unpublished). Finally, the I3 protein is a singlestranded DNA (ssDNA) binding protein (SSB) (13)(14)(15)(16), and it is the focus of the work described herein.…”

“…Like many other SSBs, the vaccinia virus I3 protein has an acidic C=-terminal tail. However, whereas this tail is thought to mediate protein-protein interactions in other cases, it has recently been shown that for I3, the C= terminus modulates the interaction of the protein with DNA (16). Truncation of the tail increases the affinity of I3 for DNA and appears to alter the structure of DNA-I3 complexes, leading to the hypothesis that the C= terminus may normally facilitate the sliding of I3 on DNA during replication.…”