# A four-in-one replicase integrating key enzymatic activities for DNA replication

## TL;DR

This study discovers a multifunctional DNA replicase in a bacteriophage that combines several key replication activities into one protein.

## Contribution

The discovery of GP55, a four-in-one replicase with unique enzymatic activities and a novel α-helix domain.

## Key findings

- GP55 integrates helicase, primase, DNA polymerase, and 3′–5′ exonuclease activities into a single protein.
- The helicase activity of GP55 is UTP/dTTP-dependent and supports strand displacement during DNA synthesis.
- A novel α-helix domain is essential for GP55's primase activity and RNA primer synthesis.

## Abstract

DNA replication is a fundamental process in all living organisms. As the most diverse and abundant biological entities on Earth, bacteriophages may utilize unconventional methods for genome replication. In this study, we identified a novel DNA replicase, GP55, from lactococcal phage 1706. GP55 comprises a helicase domain, a distinctive archaeo-eukaryotic primase domain, and a family B DNA polymerase domain, collectively exhibiting helicase, primase, and DNA polymerase activities, along with intrinsic 3′–5′ exonuclease activity. Notably, the helicase activity of GP55 is UTP/dTTP-dependent rather than ATP-dependent and facilitates strand displacement during DNA synthesis. GP55 exhibits a unique primase activity, recognizing specific but less stringent DNA sequences and preferring GTP for the initiation of RNA primer synthesis. Additionally, a newly identified α-helix domain, composed of two pairs of parallel α-helices, was found to be essential for its primase activity. The multiple activities enable GP55 to efficiently synthesize DNA de novo in the presence of dNTPs and NTPs. This study reveals a concise strategy employed by bacteriophages for genome replication using multifunctional replicases.

Graphical Abstract