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

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.

Keywords: Accessory Helicase; CMG Helicase; DNA Replication; RTEL1; Rrm3.

Figure 1. The N-terminal IDR of Rrm3 is required for Rrm3 function.

(A) Comparison of AlphaFold2-predicted structures of S. cerevisiae Rrm3 with Pif1 from Bacteroides sp 2 1 16 (BacPif1), showing that the Rrm3 Intrinsically Disordered Region (IDR) is absent from BacPif1. Root mean square deviation (RMSD) is given for the aligned helicase domains. (B) Reaction scheme for examining DNA replication termination in in vitro DNA replication reactions reconstituted with purified S. cerevisiae proteins. LRIs = Late Replication Intermediates, which result from the stalling of converging replisomes in the absence of S. cerevisiae Rrm3 or Pif1. (C) A 3189 bp plasmid template (pBS/ARS1WTA) was replicated according to the scheme in (B), with wild-type (WT) Rrm3 or Rrm3 lacking the first 193 amino acids (ΔN) added as indicated. XmaI-digested radiolabelled replication products were resolved in a native agarose gel and detected by autoradiography. (D, E) Diploid yeast cells of the indicated genotypes were sporulated and the resulting tetrads were then dissected and grown on YPD medium for 2 days at 30 °C. Source data are available online for this figure.

Figure 2. The Rrm3 IDR is necessary and sufficient for CMGE binding.

(A) The ability of Rrm3 and Rrm3ΔN to associate with CMG was monitored in the presence of Polε. The indicated factors were mixed, before immunoprecipitation of the Sld5 subunit of CMG and immunoblotting. Rrm3 and Cdc45 were detected by anti-FLAG immunoblotting in this and subsequent experiments. (B) Purified BacPif1 and a version of BacPif1 that was fused to the first 193 amino acids of the Rrm3 IDR (Rrm3N-BacPif1) visualised by SDS-PAGE and Coomassie staining. (C) The ability of BacPif1 and Rrm3N-BacPif1 to unwind a 25 bp DNA duplex, formed by annealing oligonucleotides TD254 and TD255, was monitored as described in Methods. * indicates ³²P-labelling of TD254. (D, E) The ability of Rrm3, Rrm3N-BacPif1 and BacPif1 to associate with CMG was monitored in the presence of Polε as in (A). (F) In vitro DNA replication reactions conducted as in Fig. 1B with the indicated concentrations of Rrm3N-BacPif1 and BacPif1. Source data are available online for this figure.

Figure 3. Structural modelling of Rrm3 in the budding yeast replisome.

(A, B) AlphaFold-Multimer models of Rrm3 bound to Dpb2 (A) and the GINS tetramer (B). Key interaction residues that were mutated in Rrm3 mutants (Fig. 4) are indicated. (C) Domain structure and AlphaFold2-predicted monomer structure of Rrm3. Positions of adjacent Dpb2- and Sld5-binding Short Linear Interaction Motifs (SLIMs) in the Rrm3 IDR are indicated. (D) Dpb2- and Sld5-binding SLIMs in Rrm3 were docked onto a cryo-EM structure of budding yeast CMG-Polε (PDB: 7PMK) by aligning on Dpb2 and Sld5, respectively (see Methods for more details). The disordered segment of Rrm3 that connects the two SLIMs is represented as a dashed line. The path of the excluded lagging strand DNA template, which exits the CMG central channel between Mcm3 and Mcm5, is indicated.

Figure 4. Isolation of CMGE interaction mutants in Rrm3.

(A) Schematic showing positions of mutations in Rrm3 residues 86–130, designed to disrupt binding to Polε (Rrm3Δ86-110, -CR and -6A) or GINS (Rrm3Δ111-130 and -2E). (B, C) Purified tetrameric GINS complex (B) or Polε (C) were mixed with FLAG-tagged wild-type Rrm3 or the indicated Rrm3 mutants. In (B), Rrm3-2E was included at 5, 10, 20 and 40 nM, whereas wild type and Rrm3Δ111-130 were included at 10 nM. Resultant complexes were isolated by anti-FLAG immunoprecipitation and detected by SDS-PAGE and immunoblotting. (D) The ability of wild type and the indicated mutant forms of Rrm3 to associate with CMG was monitored in the presence of Polε, as in Fig. 2A. Source data are available online for this figure.

Figure 5. CMGE binding is critical for Rrm3 function.

(A, B) In vitro DNA replication reactions conducted as in Fig. 1B with wild-type Rrm3 or the indicated Rrm3 mutants. (C, D) Diploid yeast cells of the indicated genotypes were sporulated and the resulting tetrads were then dissected and grown on YPD medium for 2 days at 30 °C. Source data are available online for this figure.

Figure 6. Structural modelling of RTEL1 in the human replisome.

(A, B) AlphaFold-Multimer models of Homo sapiens RTEL1 isoform 1 (NP_057518.1) bound to POLE2 (A) and the GINS tetramer (B). Interacting sites in RTEL1 are highlighted, and zoomed in views are shown to the right. Residue numbers relevant to RTEL1 deletion mutants (Fig. 7) are highlighted. (C) Domain structure and AlphaFold2-predicted monomer structure of RTEL1 isoform 1 (NP_057518.1). Positions of three adjacent POLE2- and GINS-binding Short Linear Interaction Motifs (SLIMs) in RTEL1 are indicated. Residues 890–1219, including the C-terminal harmonin-like (HNL1 and HNL2) domains (residues 890–975 and 1060–1140) and PCNA interaction (PIP) motif (residues 1166–1173), were removed from the AlphaFold2-predicted structure for simplicity. (D) POLE2- and GINS-binding SLIMs in RTEL1 (numbered as in (A–C)) were docked onto a cryo-EM structure of human CMG-Polε (PDB: 7PLO) by aligning on POLE2 and PSF1, respectively (see Methods for more details). Disordered segments of RTEL1 that connect the three SLIMs are represented as dashed lines. The path of the excluded lagging strand DNA template between MCM3 and MCM5 is indicated.

Figure 7. Interaction of RTEL1 with POL ε and CMG.

(A, B) Purified Homo sapiens POL ε (A) or CMG (B) were mixed with FLAG-tagged wild-type RTEL1 or the indicated RTEL1 mutants. Resultant complexes were isolated by anti-FLAG immunoprecipitation and detected by SDS-PAGE and immunoblotting. RTEL1 was detected by anti-FLAG immunoblotting. (C) Model for Rrm3 accessory helicase function during DNA replication. Dpb2- and Sld5-binding SLIMs in Rrm3 are shown in red. Removal of a protein barrier from the DNA (as an example of Rrm3 function) is depicted by a dashed line. We envisage that a similar mechanism could operate for RTEL1 in the human replisome, based on the CMGE binding mechanism we have identified. Discussed further in the text. Source data are available online for this figure.

Figure EV1. Characterisation of rrm3ΔN mutant in vitro and in vivo.

(A) Disorder prediction for S. cerevisiae Rrm3, generated using the flDPnn webserver. Residue numbers are given on the x-axis. (B) The ability of Rrm3 and Rrm3ΔN to unwind a 25 bp DNA duplex, formed by annealing oligonucleotide TD254 to TD255, was monitored as described in Methods. * indicates ³²P-labelling of TD254. (C) Similar experiments to (B) were performed three times. The percentage of unwound product was quantified in each case for reactions containing 5 nM of Rrm3, and the figure presents the mean values with standard deviations. (D, E) Diploid yeast cells of the indicated genotypes were sporulated and the resulting tetrads were then dissected and grown on YPD medium for 2 days at 30 °C.

Figure EV2. Supporting data for Fig. 2.

(A) A 3189 bp plasmid template (pBS/ARS1WTA) was replicated in the presence or absence of Rrm3 (12.5 nM) or Pif1 (5 nM) and the indicated replisome components. SmaI-digested radiolabelled replication products were resolved in a native agarose gel and detected by autoradiography. (B) Similar experiments to Fig. 2C were performed three times. The percentage of unwound product was quantified in each case, and the figure presents the mean values with standard deviations. (C) Similar experiments to Fig. 2F were performed three times. The percentage full-length products was quantified in each case, and the figure presents the mean values with standard deviations. Quantification was performed for BacPif1 and Rrm3N-BacPif1 samples that included 5 nM of each helicase.

Figure EV3. Generation and characterisation of CMGE-binding mutants of Rrm3.

(A) Purified wild-type or mutant versions of Rrm3 visualised by SDS-PAGE and Coomassie staining. * is a contaminating protein. (B, C) Purified Polε (B) or tetrameric GINS complex (C) were mixed with FLAG-tagged wild-type Rrm3 or the indicated Rrm3 mutants. Resultant complexes were isolated by anti-FLAG immunoprecipitation and detected by SDS-PAGE and immunoblotting. Rrm3 was detected by anti-FLAG immunoblotting.

Figure EV4. Supporting data showing that CMGE binding is critical for Rrm3 function.

(A, B) Similar experiments to Fig. 5A (A) and 5B (B) were performed three times. The percentage full-length products was quantified in each case, and the figure presents the mean values with standard deviations. (C, D) Diploid yeast cells of the indicated genotypes were sporulated and the resulting tetrads were then dissected and grown on YPD medium for 2 days at 30 °C.

Figure EV5. Generation and characterisation of CMGE-binding mutants of RTEL1.

(A) Wild type or mutant versions of Homo sapiens RTEL1, CMG and POL ε purified after expression in budding yeast and visualised by SDS-PAGE and Coomassie staining. * indicates a contaminant in purified RTEL1. (B, C) Purified CMG (B) or POL ε (C) were mixed with FLAG-tagged wild-type RTEL1 or the indicated RTEL1 mutants. Resultant complexes were isolated by anti-FLAG immunoprecipitation and detected by SDS-PAGE and immunoblotting. RTEL1 was detected by anti-FLAG immunoblotting.