A common mechanism for recruiting the Rrm3 and RTEL1 accessory helicases to the eukaryotic replisome

TL;DR

The study reveals how accessory helicases like Rrm3 and RTEL1 are recruited to the eukaryotic replisome to help DNA replication proceed when stalled.

Contribution

The discovery of a conserved docking mechanism via short linear interaction motifs in accessory helicases across yeast and humans.

Findings

Rrm3's N-terminal IDR contains motifs that bind CMG and Polε, positioning it near the lagging strand.

Rrm3 binding to Polε is essential for its function during DNA replication in vitro and in vivo.

RTEL1 in humans interacts with CMG and Polε in a manner similar to Rrm3, indicating a conserved mechanism.

Abstract

The eukaryotic replisome is assembled around the CMG (CDC45-MCM-GINS) replicative helicase, which encircles the leading-strand DNA template at replication forks. When CMG stalls during DNA replication termination, or at barriers such as DNA-protein crosslinks on the leading strand template, a second helicase is deployed on the lagging strand template to support replisome progression. How these ‘accessory’ helicases are targeted to the replisome to mediate barrier bypass and replication termination remains unknown. Here, by combining AlphaFold structural modelling with experimental validation, we show that the budding yeast Rrm3 accessory helicase contains two Short Linear Interaction Motifs (SLIMs) in its disordered N-terminus, which interact with CMG and the leading-strand DNA polymerase Polε on one side of the replisome. This flexible tether positions Rrm3 adjacent to the lagging strand template on which it translocates, and is critical for replication termination in vitro and Rrm3 function in vivo. The primary accessory helicase in metazoa, RTEL1, is evolutionarily unrelated to Rrm3, but binds to CMG and Polε in an analogous manner, revealing a conserved docking mechanism for accessory helicases in the eukaryotic replisome.

Accessory DNA helicases are required to overcome replisome stalling during DNA replication. This study describes a mechanism for targeting the Rrm3 accessory helicase to the budding yeast replisome via interactions with the CMG replicative helicase and DNA polymerase ε. This binding mode positions Rrm3 close to the lagging strand DNA template and is highly similar to RTEL1 in the human replisome.

Budding yeast Rrm3 contains an N-terminal Intrinsically Disordered Region (IDR) that mediates its recruitment to the replisome.The Rrm3 IDR contains adjacent motifs that interact with the CMG helicase and DNA polymerase ε, positioning Rrm3 close to the lagging strand DNA template.Rrm3 binding to DNA polymerase ε is critical for Rrm3 function during DNA replication in vitro and in vivo.The metazoan accessory helicase RTEL1 interacts with CMG and DNA polymerase ε in a highly similar manner to Rrm3.

Budding yeast Rrm3 contains an N-terminal Intrinsically Disordered Region (IDR) that mediates its recruitment to the replisome.

The Rrm3 IDR contains adjacent motifs that interact with the CMG helicase and DNA polymerase ε, positioning Rrm3 close to the lagging strand DNA template.

Rrm3 binding to DNA polymerase ε is critical for Rrm3 function during DNA replication in vitro and in vivo.

The metazoan accessory helicase RTEL1 interacts with CMG and DNA polymerase ε in a highly similar manner to Rrm3.

Short linear interaction motifs present in accessory DNA helicases from yeast and humans mediate their interaction with the CMG replicative helicase and DNA polymerase ε.

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