Substrate-selective repair and restart of replication forks by DNA translocases

Stalled replication forks are sources of genetic instability. Multiple fork-remodeling enzymes are recruited to stalled forks, but how they work to promote fork restart is poorly understood. By combining ensemble biochemical assays and single-molecule studies with magnetic tweezers, we show that SMARCAL1 branch migration and DNA-annealing activities are directed by the single-stranded DNA-binding protein RPA to selectively regress stalled replication forks caused by blockage to the leading-strand polymerase and to restore normal replication forks with a lagging-strand gap. We unveil the molecular mechanisms by which RPA enforces SMARCAL1 substrate preference. E. coli RecG acts similarly to SMARCAL1 in the presence of E. coli SSB, whereas the highly related human protein ZRANB3 has different substrate preferences. Our findings identify the important substrates of SMARCAL1 in fork repair, suggest that RecG and SMARCAL1 are functional orthologs, and provide a comprehensive model of fork repair by these DNA translocases

SMARCAL1 and WRN co-localize at stalled replication forks.

<p>(A) HeLa cells expressing GFP-WRN were treated with 2 mM HU, fixed, and stained with antibodies to SMARCAL1. (B) HeLa cells were transfected with non-targeting (NT) or WRN siRNAs then treated with 2 mM HU for the indicated times. Cells were fixed and stained with antibodies to SMARCAL1. (C) GFP-WRN expressing HeLa cells were transfected with NT or SMARCAL1 siRNAs, treated with 2 mM HU for the indicated times, fixed, and imaged for WRN. No significant difference is observed between the NT and SMARCAL1 siRNA samples.</p

Identification of SMARCAL1 interacting proteins by mass spectrometry.

<p>(A) 293T cells were transfected with a Flag-SMARCAL1 expression vector or an empty vector control. Cells were harvested, lysed and Flag antibody conjugated beads were used to immunopurify SMARCAL1. HeLa cells expressing endogenous SMARCAL1 were harvested, lysed, and SMARCAL1-909 antibody or IgG control antibody were used for immunopurification. Proteins were eluted from beads with either the Flag or SMARCAL1-909 peptides and subjected to 2D-LC-tandem mass spectrometry. Where indicated, the cells were treated with 2 mM HU for 16 h prior to harvesting. The number of peptides identified in each purification is reported. (B–D) HeLa nuclear extracts were prepared. Control IgG, SMARCAL1-909 antibody, or WRN antibody immunoprecipitates (IP) as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Where indicated the nuclear extracts were treated with benzonase prior to the immunoprecipitation. In (D) the cells were either mock treated (Unt.) or incubated with 2 mM HU for 16 h prior to harvesting.</p

SMARCAL1 and WRN work independently to catalyze fork regression.

<p>Purified SMARCAL1 and WRN proteins were incubated with a ³²P-labeled model replication fork substrate capable of undergoing fork regression (see supplemental table S1). The substrate contains a 30-nucleotide ssDNA gap on the leading strand. RPA was pre-bound to the substrate prior to the addition of enzymes. (A) 500pM SMARCAL1 was used where indicated. The Ctl reactions lacked WRN protein to show the amount of spontaneous fork regression without SMARCAL1 or the amount of fork regression in the presence of only SMARCAL1. R764Q indicates reactions with the ATPase deficient SMARCAL1 R764Q mutant as a control. (B) Quantitation of the percent of product formed in each reaction. The amount of spontaneous branch migration in the absence of any added protein was subtracted from each sample.</p

RPA acts as a scaffold to mediate the SMARCAL1-WRN interaction.

<p>(A) GST-WRN proteins bound to glutathione beads were incubated with HeLa nuclear extracts. After extensive washing, bound proteins were separated by SDS-PAGE and immunoblotted with antibodies to SMARCAL1 or RPA32. A coomassie stained gel was also prepared to document the amount of GST proteins added to the lysates. (B) Recombinant RPA, SMARCAL1, or both proteins were incubated with GST-WRN fragments bound to glutathione beads. After washing, bound proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (C) Flag immunoprecipitates from 293T cell lysates after transfection with Flag-SMARCAL1 wild-type (WT) or mutant expression vectors were separated by SDS-PAGE and immunoblotted with the indicated antibodies.</p

DNA replication stress triggers rapid DNA replication fork breakage by Artemis and XPF.

DNA replication stress (DRS) leads to the accumulation of stalled DNA replication forks leaving a fraction of genomic loci incompletely replicated, a source of chromosomal rearrangements during their partition in mitosis. MUS81 is known to limit the occurrence of chromosomal instability by processing these unresolved loci during mitosis. Here, we unveil that the endonucleases ARTEMIS and XPF-ERCC1 can also induce stalled DNA replication forks cleavage through non-epistatic pathways all along S and G2 phases of the cell cycle. We also showed that both nucleases are recruited to chromatin to promote replication fork restart. Finally, we found that rapid chromosomal breakage controlled by ARTEMIS and XPF is important to prevent mitotic segregation defects. Collectively, these results reveal that Rapid Replication Fork Breakage (RRFB) mediated by ARTEMIS and XPF in response to DRS contributes to DNA replication efficiency and limit chromosomal instability

Substrate-selective repair and restart of replication forks by DNA translocases

Stalled replication forks are sources of genetic instability. Multiple fork-remodeling enzymes are recruited to stalled forks, but how they work to promote fork restart is poorly understood. By combining ensemble biochemical assays and single-molecule studies with magnetic tweezers, we show that SMARCAL1 branch migration and DNA-annealing activities are directed by the single-stranded DNA-binding protein RPA to selectively regress stalled replication forks caused by blockage to the leading-strand polymerase and to restore normal replication forks with a lagging-strand gap. We unveil the molecular mechanisms by which RPA enforces SMARCAL1 substrate preference. E. coli RecG acts similarly to SMARCAL1 in the presence of E. coli SSB, whereas the highly related human protein ZRANB3 has different substrate preferences. Our findings identify the important substrates of SMARCAL1 in fork repair, suggest that RecG and SMARCAL1 are functional orthologs, and provide a comprehensive model of fork repair by these DNA translocases

Eprinomectin resistance in Haemonchus contortus: Phenotype, Genomic and Transcriptomic

International audienceHaemonchus contortus is a gastrointestinal nematode parasite of small ruminants causing haemonchosis characterized by anemia that can lead to death. Highly pathogenic, H. contortus causes major economic losses on farms and affects animal welfare. Control of this parasite relies essentially on the use of anthelmintics from the macrocyclic lactone (ML) family. In dairy sheep farms Eprinomectin (EPR) is the only ML used to treat haemonchosis because it is the only null milk withdrawal anthelmintic molecule currently available. EPR intensive use has led to the recent appearance of cases of resistance that are jeopardizing the control of haemonchosis. Hence, we recently reported eprinomectin-resistant isolates of Haemonchus contortus in 5 dairy sheep farms from the Pyrénées Atlantiques (Jouffroy, S. et al. Parasitology (2023). EPR resistance mechanisms are poorly understood in parasitic nematodes. By combining farm Anthelmintic treatment history with phenotypic, genomic and transcriptomic data, we aim at deciphering the determinant of Eprinomectin resistance and proposing resistant markers. These approaches enabled us to unveil the very high resistance to Eprinomectin of Haemonchus contortus isolates (&gt;30x), but also that the resistant populations selected a narrow Quantitative Trait Locus (QTL) on chromosome V and genes expressed in neuronal tissues, associated with anthelmintic response and implicated in diverse cellular and metabolic functions. Investigations of individual genes deregulated and present on chromosome-V QTL are ongoing.</div