# Structures of nucleotide-bound Redondovirus Rep protein link conformation and function

## TL;DR

This paper reveals the structure of a Rep protein from a human-associated virus, showing how it changes shape to perform DNA replication tasks.

## Contribution

The study presents the first full-length structure of a Redondovirus Rep protein in a dodecameric assembly, linking conformational changes to its replication functions.

## Key findings

- Cryo-EM structures show Rep in ATP-bound and ADP-bound states, revealing conformational differences.
- A dodecameric Rep assembly was identified, with ordered helicase and endonuclease domains.
- Conserved residues suggest the dodecamer may be functionally important for many CRESS-DNA viruses.

## Abstract

Circular Rep-encoding single-stranded DNA (CRESS-DNA) virus Rep proteins are multidomain enzymes that mediate viral DNA rolling-circle replication. Reps nick viral DNA to expose a 3’ end for polymerase extension, provide an NTP-dependent helicase activity for DNA unwinding, and join nicked ends to form circular viral genomes. Here, we present the first structures of a Rep protein from the Redondoviridae family, a newly discovered family of human-associated CRESS-DNA viruses that replicates within the oral protozoan Entamoeba gingivalis. Using cryo-EM, we characterized the hexameric structures of a Redondovirus Rep helicase bound with ATPγS, representing the initial ATP-bound state, and with ADP, reflecting the protein state after hydrolysis. The ADP state, but not the ATP state of Rep shows a staircase arrangement of DNA-binding loops that plays a central role in current models for SF3 helicase function. Additionally, we determined a head-to-tail dodecameric structure of ATPγS-bound Rep, in which both the helicase and endonuclease domains are ordered. Conservation of residues involved in stabilizing the dodecamer suggest that this assembly may be functionally relevant for many CRESS-DNA viruses. The positioning of endonuclease domains in the Rep hexamer, combined with our biophysical analyses of Rep oligomerization, provide new insights into Rep function during viral replication.

We have used cryo-electron microscopy and solution biophysical techniques to understand the structure of the Rep protein from a Redondovirus, a CRESS DNA virus originally found in human respiratory samples. The small circular Rep-encoding single-stranded (CRESS) DNA viruses produce only two known proteins; one forms the viral capsid, and the other, called Rep, assists in replicating the genome. Rep has at least three distinct biochemical activities. It cleaves the genome to generate a single-strand break, unwinds the double-stranded DNA generated during replication by the host cell’s machinery, and rejoins the break to form a single-stranded DNA circle that is packaged into a new viral particle. Previous studies of Rep have revealed the structures of the isolated DNA-unwinding (helicase) and cleaving/rejoining (endonuclease) domains, but the structure of an entire Rep protein has been elusive. Here, we describe the structure of full-length Redondovirus Rep in an unexpected dodecameric assembly. Together with structures of the helicase and endonuclease domains, the models provide new insights into how Rep might coordinate its cleavage, unwinding, and rejoining activities during the viral replication cycle.