A bifunctional RNase H protein supports DNA replication and transcription in nucleus-forming Chimalliviridae phages
- 1University of California San Diego; Howard Hughes Medical Institute
Abstract
Jumbo bacteriophages of the Chimalliviridae family replicate their genomes within a proteinaceous phage nucleus that spatially segregates viral DNA replication and transcription from translation. While the phage nucleus protects viral DNA from host defenses, it also imposes unique constraints on genome replication and gene expression. Here we identify the phage-encoded protein Rhc (RNase H-clamp) as a central mediator of genome transactions within the phage nucleus. Using the Escherichia coli infecting bacteriophage Goslar, we show that Rhc functions as an essential component of a non-canonical three-subunit DNA polymerase complex composed of DNAPN, DNAPC and Rhc. Rhc physically interacts with the DNAPC subunit, and both the catalytic activity of Rhc's N-terminal RNase H domain and its C-terminal sliding clamp-derived DNA binding domain are required for genome replication, nuclear maturation, and productive infection. Further, Goslar Rhc also supports transcription within the phage nucleus through a function that is separable from its DNA replication function. Rhc depletion results in reduced transcription of a subset of late genes, in a pattern that suggests a role for Rhc in resolving R-loops. Imaging and cryo-electron tomography reveal that loss of Rhc arrests phage development at a nascent nuclear stage, uncoupling nuclear assembly from maturation. Together, these findings identify Rhc as a multifunctional nuclear factor that mediates both DNA replication and transcription in nucleus-forming jumbo phages, revealing how viruses repurpose a conserved enzymatic activity to coordinate gene expression within a compartmentalized replication environment.
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BibTeX
@misc{rani2026bifunctional,
title = {{A bifunctional RNase H protein supports DNA replication and transcription in nucleus-forming Chimalliviridae phages}},
author = {P. Rani and N. Klusch and S. Barret and T. Forman and M. Levine and M. Riggi and C. Rees and R. Liu and L. Hoffman and M. Norquist and D. Basu and K. Inlow and E. G. Armbruster and C. J. Morgan and J. Pogliano and K. D. Corbett and E. Villa},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.01.755736},
url = {https://www.biorxiv.org/content/10.64898/2026.10.01.755736v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - A bifunctional RNase H protein supports DNA replication and transcription in nucleus-forming Chimalliviridae phages
AU - P. Rani
AU - N. Klusch
AU - S. Barret
AU - T. Forman
AU - M. Levine
AU - M. Riggi
AU - C. Rees
AU - R. Liu
AU - L. Hoffman
AU - M. Norquist
AU - D. Basu
AU - K. Inlow
AU - E. G. Armbruster
AU - C. J. Morgan
AU - J. Pogliano
AU - K. D. Corbett
AU - E. Villa
PY - 2026
DA - 2026/10/01
PB - bioRxiv
DO - 10.64898/2026.10.01.755736
UR - https://www.biorxiv.org/content/10.64898/2026.10.01.755736v1
AB - Jumbo bacteriophages of the Chimalliviridae family replicate their genomes within a proteinaceous phage nucleus that spatially segregates viral DNA replication and transcription from translation. While the phage nucleus protects viral DNA from host defenses, it also imposes unique constraints on genome replication and gene expression. Here we identify the phage-encoded protein Rhc (RNase H-clamp) as a central mediator of genome transactions within the phage nucleus. Using the Escherichia coli infecting bacteriophage Goslar, we show that Rhc functions as an essential component of a non-canonical three-subunit DNA polymerase complex composed of DNAPN, DNAPC and Rhc. Rhc physically interacts with the DNAPC subunit, and both the catalytic activity of Rhc's N-terminal RNase H domain and its C-terminal sliding clamp-derived DNA binding domain are required for genome replication, nuclear maturation, and productive infection. Further, Goslar Rhc also supports transcription within the phage nucleus through a function that is separable from its DNA replication function. Rhc depletion results in reduced transcription of a subset of late genes, in a pattern that suggests a role for Rhc in resolving R-loops. Imaging and cryo-electron tomography reveal that loss of Rhc arrests phage development at a nascent nuclear stage, uncoupling nuclear assembly from maturation. Together, these findings identify Rhc as a multifunctional nuclear factor that mediates both DNA replication and transcription in nucleus-forming jumbo phages, revealing how viruses repurpose a conserved enzymatic activity to coordinate gene expression within a compartmentalized replication environment.
ER -