Rrm3 and Pif1 division of labor during replication through leading and lagging strand G-quadruplex

Abstract Members of the conserved Pif1 family of 5′-3′ DNA helicases can unwind G4s and mitigate their negative impact on genome stability. In Saccharomyces cerevisiae, two Pif1 family members, Pif1 and Rrm3, contribute to the suppression of genomic instability at diverse regions including telomeres, centromeres and tRNA genes. While Pif1 can resolve lagging strand G4s in vivo, little is known regarding Rrm3 function at G4s and its cooperation with Pif1 for G4 replication. Here, we monitored replication through G4 sequences in real time to show that Rrm3 is essential for efficient replisome progression through G4s located on the leading strand template, but not on the lagging strand. We found that Rrm3 importance for replication through G4s is dependent on its catalytic activity and its N-terminal unstructured region. Overall, we show that Rrm3 and Pif1 exhibit a division of labor that enables robust replication fork progression through leading and lagging strand G4s, respectively.


Figure S1:
Replication times through Glutamate tRNA (YNCI0011W) located between the lacO and tetO arrays in a head-on (HO) orientation, measured for cells expressing Rrm3 (blue) or for cells depleted of Rrm3 (orange).Replication through the tRNA-Glu is significantly slowed down upon Rrm3 depletion in agreement with previous studies showing replication stalling at tRNAs in rrm3-deleted strains (see text for details).The Glutamate tRNA strain contains RRM3-AID and Rrm3 depletion was induced by the addition of 1mM IAA, as described in the Materials and Methods section.Detection of Rrm3-AID depletion was performed using anti-FLAG antibody just before (0 h) or 1 h following IAA (auxin) addition (1 mM of IAA) to the media (top).Detection of the complemented Rrm3-6xHA variants, expressed from the URA3 locus, was performed using anti-HA antibodies.Longer exposure of the membrane was performed to detect the rrm3-∆54 variant that is expressed at a low level (middle).Detection of Pgk1 using anti-Pgk1 antibody was performed as a loading control (bottom).For details regarding procedure and antibodies, see Materials and Methods section.

Figure S2 :
Figure S2: Representative results of single cell analysis showing replication times of ~30 Kb (distance between mid-lacO and mid-tetO arrays) in cells containing G4(A+B) between the arrays.(A-B) Cells from WT strain containing leading G4(A+B) in the presence of Rrm3 (A) or in the absence of Rrm3 (B).(C-D) representative cells from WT (C) and pif1-deletion (D) strains containing lagging G4(A+B).Solid lines represent a fit of the data to a sigmoidal function, green and red mid-points are indicated by dashed lines.Replication time for each cell is shown as the difference in midpoints of the sigmoidal fit between the red (tetO) and green (lacO) channels.Strains of panels A-B contain RRM3-AID, and Rrm3 depletion was induced by the addition of 1mM IAA, as described in the Materials and Methods section.

Figure S3 :
Figure S3: Replication times through a region of ∼30 kb containing G4(A+B) located on the leading or lagging strand templates, measured for WT (blue) or pif1-deletion strains (orange).Replication times in the absence of G4s for the two strains is shown for comparison.Longer replication times through lagging G4(A+B) in pif1-deletion strain, relative to WT strain, indicate slower fork progression through the G4 sequence.No significant replication slowdown is observed for replication through leading G4(A+B) in the pif1-deletion strain, relative to WT strain.

Fig. S4 :
Fig. S4: Synchronization and release of strains containing RRM3-AID (A) or RRM3-AID and pif1deletion strain (B) and leading G4(A+B) located 3 kb from ARS413.Samples for flow cytometry analysis were taken from asynchronized population (Asy) following 2:30 h of synchronization with alpha factor (G1) and at different time points (30, 47, 60 and 90 min) following release into S phase.

Fig. S5 :
Fig. S5: Copy number variation experiments in RRM3-AID and pif1-deletion strain containing leading G4(A+B) following cell release into S-phase, indicating a decrease in copy number downstream of G4(A+B)

Figure S7 :
Figure S7: (A)A structural model of Rrm3 generated by the alpha-fold server (https://alphafold.ebi.ac.uk/).The N-terminal region of Rrm3 composed of residues 1-230 is unstructured (cyan).In contrast, residues 230-723 of Rrm3 are well structured containing -helical and -strands forming the helicase domain (green).(B) Prediction of intrinsically disordered regions in Rrm3 using the IUPred3 algorithm.The score of prediction (0-1) is plotted against Rrm3 residue number.The threshold of intrinsically disordered region detection is 0.5 and residues with a higher score are considered to be disordered.In accordance with the alpha-fold Rrm3 structural prediction (A) residue 1-230 are predicted to be disordered.

Figure S8 :
Figure S8: Western blot analysis for the detection of Rrm3-AID depletion following auxin (IAA) addition and the complementation of cells with WT Rrm3 and different N-terminal truncated variants of Rrm3 expressed from the native RRM3 promoter.The different N-terminal truncated Rrm3 variants include rrm3-∆54, rrm3-∆186, rrm3-∆212 and rrm3-∆230.Detection of Rrm3-AID depletion was performed using anti-FLAG antibody just before (0 h) or 1 h following IAA (auxin) addition (1 mM of IAA) to the media (top).Detection of the complemented Rrm3-6xHA variants, expressed from the URA3 locus, was performed using anti-HA antibodies.Longer exposure of the membrane was performed to detect the rrm3-∆54 variant that is expressed at a low level (middle).Detection of Pgk1 using anti-Pgk1 antibody was performed as a loading control (bottom).For details regarding procedure and antibodies, see Materials and Methods section.