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Hrq1/RECQL4 regulation is critical for preventing aberrant recombination during DNA intrastrand crosslink repair and is upregulated in breast cancer [1]

['Thong T. Luong', 'University Of Pittsburgh School Of Medicine', 'Department Of Pharmacology', 'Chemical Biology', 'Upmc Hillman Cancer Center', 'Pittsburgh', 'Pennsylvania', 'United States Of America', 'Zheqi Li', 'Women S Cancer Research Center']

Date: 2022-11 

Human RECQL4 is a member of the RecQ family of DNA helicases and functions during DNA replication and repair. RECQL4 mutations are associated with developmental defects and cancer. Although RECQL4 mutations lead to disease, RECQL4 overexpression is also observed in cancer, including breast and prostate. Thus, tight regulation of RECQL4 protein levels is crucial for genome stability. Because mammalian RECQL4 is essential, how cells regulate RECQL4 protein levels is largely unknown. Utilizing budding yeast, we investigated the RECQL4 homolog, HRQ1, during DNA crosslink repair. We find that Hrq1 functions in the error-free template switching pathway to mediate DNA intrastrand crosslink repair. Although Hrq1 mediates repair of cisplatin-induced lesions, it is paradoxically degraded by the proteasome following cisplatin treatment. By identifying the targeted lysine residues, we show that preventing Hrq1 degradation results in increased recombination and mutagenesis. Like yeast, human RECQL4 is similarly degraded upon exposure to crosslinking agents. Furthermore, over-expression of RECQL4 results in increased RAD51 foci, which is dependent on its helicase activity. Using bioinformatic analysis, we observe that RECQL4 overexpression correlates with increased recombination and mutations. Overall, our study uncovers a role for Hrq1/RECQL4 in DNA intrastrand crosslink repair and provides further insight how misregulation of RECQL4 can promote genomic instability, a cancer hallmark.

RECQL4 is a DNA helicase and functions during DNA replication and repair. While loss-of-function RECQL4 mutations are found in diseases characterized by developmental defects and cancer, such as Rothmund-Thomson syndrome, over-expression of RECQL4 is also observed in cancer, such as breast cancer. Therefore, RECQL4 protein expression must be tightly regulated. Here we used the budding yeast homolog of RECQL4, Hrq1, and discovered that overexpression of Hrq1 protein levels result in increased recombination and mutations, both cancer hallmarks. We find that Hrq1 functions to mediate repair of a specific type of DNA damage, intrastrand crosslinks, which occur when DNA nucleotides on the same strand are chemically linked together. These findings are also conserved in humans suggesting a common mechanism between yeast Hrq1 and human RECQL4. Overall, our study identifies a conserved role for RECQL4 in DNA intrastrand crosslink repair and provides insights into how its misregulation could promote cancer development.

Funding: This study was supported by grants from the National Institutes of Health (ES030335 to K.A.B.) and the Department of Defense (BC201356 to K.A.B.). This work was also supported by the Hillman Fellows for Innovative Cancer Research Program to K.A.B. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Luong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here, similar to plants and in contrast to previous studies in budding and fission yeast, we find that the budding yeast Hrq1 has a role in the error-free branch of PRR specifically during intrastrand crosslink repair. Furthermore, we performed genetic analysis that suggests that human RECQL4 also functions during PRR. Paradoxically, despite a conserved role in cisplatin resistance, Hrq1 is degraded by the proteasome following cisplatin exposure. We find that HRQ1 overexpression or its stabilization leads to increased recombination and mutation rates. Furthermore, suggesting a conserved regulatory mechanism, we show that endogenous human RECQL4 protein levels decrease following cisplatin and acetaldehyde treatment. Moreover, overexpression of RECQL4 results in increased RAD51 foci, which is in part dependent on its helicase activity. Lastly, bioinformatic analysis reveals that high levels of RECQL4 are correlated with increased tumor mutation burden. Importantly, we observe that in TNBC, the protein levels of RECQL4 are a predictive marker to cisplatin response. Our work uncovers a role for Hrq1 in the error-free branch PRR repair and a conserved regulatory mechanism between yeast Hrq1 and mammalian RECQL4 following DNA intrastrand crosslink damage.

Recent studies demonstrate that Hrq1 functions during NER to repair cisplatin-induced DNA lesions [ 44 – 47 ]. However, there are conflicting studies as to whether Hrq1 also has a role in PRR [ 44 – 46 ]. For example, a study in A. thaliana suggests that Hrq1 functions in PRR while two independent studies in S. pombe and S. cerevisiae, respectively, suggest that Hrq1 functions independently of PRR [ 44 – 46 ]. Therefore, it remains controversial as to whether Hrq1 or its mammalian homology, RECQL4, truly functions during PRR.

DNA crosslinking agents can induce two types of damage including, interstrand crosslinks (ICL) and intrastrand crosslinks. In ICLs, the Watson and Crick strands are covalently linked, whereas in an intrastrand crosslink, the same DNA strand is covalently linked to itself [ 23 – 26 ]. Due to the different nature of the crosslinks, the mechanism of how these two adducts are repaired is also unique. While there are some key differences in how ICLs are repaired from yeast to man, during replication-coupled ICL repair, the steps are largely conserved [ 27 – 31 ]. Following damage recognition, endonucleases nick either side of the damaged DNA, which then mediates exonuclease to come in and degrade the damaged DNA. This “unhooking” step results in a ssDNA gap. This gap may be filled in by the post-replicative repair (PRR) pathway [ 28 , 32 – 35 ]. The PRR pathway consists of an error-prone and error-free branch. Utilization of the different branches is mediated by PCNA (Pol30 in yeast) ubiquitylation. For example, in both yeast and humans mono-ubiquitylation of PCNA at lysine 164 (K164) recruits error-prone translesion synthesis polymerases to fill the gap [ 36 ]. On the other hand, polyubiquitylation of the same K164 residue on PCNA results in error-free homology-directed repair [ 37 ]. In contrast to ICLs, intrastrand crosslinks are primarily repaired by the nucleotide excision repair (NER) pathway [ 38 – 41 ]. However, if the replication fork encounters the intrastrand crosslink and stalls, then the PRR and DNA damage tolerance pathways can mediate bypass of this adduct using the mechanism described above [ 36 , 37 , 42 , 43 ]. Subsequently NER, will excise and degrade the damaged DNA and the gap with be filled using DNA polymerases and ligases.

Although RECQL4 is critical for genome integrity and disease prevention, previous studies of mammalian RECQL4 were stymied due to technical difficulties, including the embryonic lethality of mouse knockout models and the inviability of human RECQL4 knockout cell lines [ 8 , 17 – 19 ]. In 2008, HRQ1, was discovered as the Saccharomyces cerevisiae homolog of RECQL4 [ 20 ]. Since HRQ1 is non-essential, yeast is a valuable model to elucidate RECQL4 gene family function. For example, analysis of hrq1 Δ cells identified a novel function for Hrq1 in DNA crosslink repair [ 21 , 22 ]. HRQ1-disrupted cells are sensitive to DNA crosslinking agents such as cisplatin and mitomycin C (MMC), which predominantly create intrastrand or interstrand crosslinks, respectively. Furthermore, recent studies suggest that human RECQL4 also has a role in crosslink repair since RECQL4 shRNA knockdown leads to cisplatin sensitivity in a triple-negative breast cancer (TNBC) cell line [ 14 ]. Although the RECQL4 gene family has a conserved role in crosslink repair, its molecular function during this process has yet to be elucidated.

Accurate and timely repair of DNA damage is critical for genomic integrity and human health. Disruptions of DNA repair genes are frequently associated with diseases such as cancer and aging. One such gene is RECQL4, which belongs to the evolutionarily conserved family of RecQ helicases. This family of 3’ to 5’- DNA helicases is often referred to as the “Guardians of the Genome” due to crucial roles in DNA recombination, replication, and repair that are conserved from yeast to man [ 1 – 5 ]. Mutations in RECQL4 are associated with three heritable autosomal diseases: Rothmund-Thomson syndrome (RTS type II), Baller-Gerold syndrome (BGS), and RAPADILINO, each characterized by developmental defects, cancer and/or premature aging [ 6 – 12 ]. Although RECQL4 dysfunction is associated with hereditable diseases, recent studies have shown that overexpression of RECQL4 is linked to multiple cancer types such as breast, hepatic, gastric, and prostate. In each cancer type, high levels of RECQL4 are correlated with poor prognosis [ 13 – 16 ]. Since both inactivation or overexpression of RECQL4 cause human disease and genetic instability, RECQL4 protein levels must be tightly regulated.

Results

Hrq1 is degraded by the proteasome upon cisplatin treatment Previous reports demonstrate that Hrq1 is important during DNA crosslink repair [21,22]. Consistent with prior studies [21,22], we observe that hrq1Δ cells are cisplatin sensitive compared to wild-type (WT) cells (Fig 1A). Since Hrq1 is needed for resistance to DNA crosslinking agents, we reasoned that Hrq1 protein levels may increase upon cisplatin exposure. To address this, we 9xMyc tagged Hrq1 on its C-terminus at its endogenous locus and promoter. We verified that 9xMyc-Hrq1 was functional upon exposure of the HRQ1-9myc yeast strain to cisplatin by comparing its growth to the parental WT strain (S1A Fig). Although Hrq1 is needed for cisplatin resistance, we paradoxically observe that Hrq1 steady state levels significantly decrease upon cisplatin exposure (Fig 1B). Importantly, Hrq1 degradation is specific to cisplatin since other types of DNA damage, such as methyl methanesulfonate (MMS; an alkylating agent), ionizing radiation (IR; which induces double-strand breaks), or hydroxyurea (HU; which depletes dNTP pools), have no detectible effect on Hrq1 steady state levels (Fig 1B). To verify these results, we repeated these experiments with multiple doses of MMS, IR, and HU and we observe that Hrq1 protein levels still remained unchanged (S1B Fig). These results suggest that the reduced Hrq1 protein levels observed are specific to cisplatin. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Although Hrq1 is needed for cisplatin resistance, it is degraded by the proteasome upon cisplatin exposure. (A) HRQ1-null cells are sensitive to cisplatin. Wild-type (WT) or hrq1Δ disrupted cells were five-fold serially diluted on medium containing 30 μg/ml cisplatin and/or 0.1% DMSO, grown for 48 hours at 30°C, and photographed. (B) Hrq1 level is stable following treatment with other DNA damaging agents: cisplatin (100 ug/ml), MMS (0.03%), IR (100 Gy), HU (100 mM). Exponentially growing cells with Hrq1-9xMYC were treated with the indicated drugs for 2 hours before being harvested for western. (C) Hrq1 protein levels are decreased upon cisplatin treatment. Exponentially growing cells with Hrq1-9xMYC were incubated with cycloheximide in the presence or absence of 100 μg/ml cisplatin and/or 0.1% DMSO. Quantification of the proportion of Hrq1 remaining relative to time 0 (before CHX addition) and the loading control, Kar2. The experiment was performed five times with mean and standard error plotted (Raw densitometry data in Sheets A-E in S1 Data). It is important to note that Hrq1 and the loading control, Kar2, were analyzed from the same gels to account for pipetting errors. Since Hrq1 is not as abundant as the loading control, there is a limitation for the densitometry analysis. (D) The proteasome degrades Hrq1 following cisplatin exposure. PDR5 disrupted cells were untreated (0.1% DMSO), cisplatin treated, or pretreated for one hour with 50 μM MG-132 before cisplatin addition with 0.1% DMSO. Cycloheximide chases were performed the similarly as (B) but further timepoints were taken. https://doi.org/10.1371/journal.pgen.1010122.g001 Next, we sought to determine if the reduced Hrq1 protein levels observed in cisplatin-treated cells are due to protein degradation. To measure Hrq1 protein stability, we performed cycloheximide chase experiments and analyzed cells for Hrq1 protein every 20 minutes for 120 minutes following cycloheximide addition (Fig 1C). Consistent with our previous observations, cisplatin treatment led to a decrease in Hrq1 protein levels, where approximately 50% of Hrq1 remains 30 minutes following cycloheximide addition, whereas in DMSO treatment more than 50% of Hrq1 still remains at 120-minute mark (Fig 1C). Proteasomal degradation is an important regulatory mechanism to ensure proper and timely DNA repair for many DNA repair proteins [48]. Therefore, one possibility is that Hrq1 protein levels are regulated by the 26S ubiquitin proteasome system (UPS) following DNA damage. To determine whether Hrq1 is degraded by the UPS following cisplatin treatment, we performed cycloheximide chase experiments in the presence of the proteasome inhibitor, MG-132 (Fig 1D). Hrq1 protein levels were stabilized following cisplatin exposure when the UPS is inhibited (Fig 1D). Note that in order to keep the intracellular concentration of MG-132 high, the drug efflux pump, PDR5, is also disrupted [49]. These results suggest that Hrq1 is marked for proteasomal degradation.

The E3 ubiquitin ligase, Rad16, target Hrq1 for degradation following cisplatin Since Hrq1 protein levels may be proteasome regulated, we sought to identify the E3 ubiquitin ligase that targets Hrq1. In S. cerevisiae, there are 60–100 E3 enzymes, so we prioritized E3 enzymes known to regulate DNA damage response proteins. For example, the NER gene, Rad4 (mammalian XPC), functions upstream of Hrq1 during crosslink repair, and its protein levels are regulated by the UPS following DNA damage [21,50]. Therefore, one possibility is that Rad4 and Hrq1 are targeted by the same E3 enzyme, Rad16 [51,52]. Thus, we examined whether Rad16 regulates Hrq1 protein levels. Indeed, we find that Hrq1 protein levels are largely stabilized in rad16Δ cells in cisplatin treated conditions (Fig 2A). Therefore, loss of RAD16 E3 ubiquitin ligase results in Hrq1 protein stabilization following cisplatin exposure. Note that although deletion of RAD16 stabilizes Hrq1 following cisplatin exposure, in untreated conditions Hrq1 levels still decrease. Therefore, there are likely additional E3 ubiquitin ligases that regulate Hrq1 protein levels independent of its role in the DNA damage response. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Hrq1 protein levels are stabilized in the absence of the E3 Ub-ligase, RAD16. (A) Deletion of Rad16 stabilizes Hrq1 following cisplatin exposure. Hrq1-9xMYC expressing wild-type (WT) or rad16Δ, cells were incubated with cycloheximide in the presence or absence of 100 μg/ml cisplatin and/or 0.1% DMSO. Note blot from Fig 1C was reshown for comparison. (B) Quantification of the proportion of Hrq1 remaining relative to time 0 (before CHX addition) and the loading control, Kar2, are plotted on the graph in log scale from WT and rad16Δ cells. Each experiment was performed three to 5 times with standard error plotted (Raw densitometry data in Sheets F-H in S1 Data). Note that the WT cisplatin treated time course is replotted from Fig 1C, for direct comparison to rad16Δ cisplatin treated cells. https://doi.org/10.1371/journal.pgen.1010122.g002 We also examined potential E2 enzymes that regulate Hrq1. In yeast, there are only thirteen known E2s. Of the E2 enzymes only two, Ubc13 and Rad6, are associated with the DNA damage response [53]. To test whether either of these two genes may regulate Hrq1, we knocked out either RAD6 or UBC13 in a Hrq1-9myc tagged strain and performed cycloheximide chase experiments. Disruption of either UBC13 or RAD6 only led to a mild stabilization of Hrq1 (S2A and S2B Fig). It is possible that in the absence of either UBC13 or RAD6, that the other E2 enzyme could compensate for each other. To test this hypothesis, we created a double ubc13Δ rad6Δ knockout and analyzed Hrq1 protein levels by cycloheximide chase. We observe that deletion of both UBC13 and RAD6 does not fully stabilize Hrq1 protein levels (S2C Fig). These results suggest UBC13 and RAD6 do not compensate for each other and there are likely additional E2s that regulate Hrq1 protein levels.

Hrq1 functions during error-free PRR As stated above, DNA crosslinks are repaired using different pathways depending upon the cell cycle stage. For example, during replication, DNA intrastrand crosslinks are bypassed by the PRR pathway and then subsequently repaired by NER. Therefore, to determine whether Hrq1 functions during a specific cell cycle stage, we asked whether its protein levels are cell cycle regulated. We synchronized Hrq1-9xMYC expressing cells in G1 with alpha factor and released them into fresh medium to enable cell cycle progression. Protein extracts were made from equal cell numbers every 20 minutes and Hrq1 protein levels analyzed and compared to Kar2 as a loading control and Clb2 as a G2/M regulated cyclin. Consistent with a role during replication, Hrq1 protein levels begin to increase in S/G2 at 40 min after alpha factor release and then plateaus whereas Clb2 protein peaks starting at 60 min (Fig 3A). Cell cycle progression was confirmed by FACS analysis (Fig 3A). As Hrq1’s expression is increased during S/G2, this suggests that it may have a role in during replication where PRR also functions. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Hrq1 functions during error-free post-replicative repair. (A) Hrq1 expression increases during S/G2 and then plateaus. Hrq1-9xMYC expressing cells were either untreated (asynchronous, AS) or cell cycle arrested in G1 with α-factor. The α-factor arrested cells were subsequently released into fresh YPD medium (0 min) and grown for 120 min. Protein samples from the indicated time points were analyzed by western blot for Hrq1 (anti-MYC), the G2/M cyclin, Clb2, (anti-Clb2), or a loading control, Kar2 (anti-Kar2). Quantification from three experiments, the mean with SEM is shown (Raw densitometry data in S2 Data). The cell cycle stage was analyzed FACS. (B) Schematic of post-replicative repair pathways. When the replication fork stalls, Pol30 (yellow trimer) is initially monoubiquitylated (Ub, purple circle) via Rad6-Rad18 (light and dark blue circles) on lysine 164 (K164). Monoubiquitylation of PCNA recruits the error-prone translesion synthesis polymerases, i.e. Rev1 (gray circle) to bypass the lesion. Alternatively, PCNA is further poly-ubiquitylated by Rad5-Ubc13-Mms2 (light, medium, and dark purple complex). Polyubiquitylation of PCNA mediates error-free repair through template switching, which is a homolog directed process. A bypass intermediate is shown with the newly synthesized DNA in red and the lesion as a yellow star. (C, D) Hrq1 functions in the same pathway as Rad6 and Pol30. The indicated yeast strains were five-fold serial diluted onto SC medium containing DMSO and/or SC medium containing the indicated amount of cisplatin. The plates were photographed after 2 days of incubation at 30°C in the dark. (E) Hrq1 functions in the same pathway as Rad5 and Ubc13. Serial dilutions of the indicated yeast strains were performed as described in (C). (F) Hrq1 functions in a different pathway than Rev1. Serial dilutions of the indicated yeast strains were performed as described in (C). https://doi.org/10.1371/journal.pgen.1010122.g003 Previous studies in plants, fission yeast, and budding yeast have confounding results as to whether Hrq1 functions during PRR [44–46]. Therefore, it remains controversial as to the role of Hrq1 during crosslink repair. To determine if Hrq1 has a role in PRR, we carefully and systematically examined the genetic relationship between HRQ1 and multiple members of the PRR pathway. We combined HRQ1 mutants with disruption of PRR genes and tested these mutants for increased cisplatin sensitivity. Since Rad6/Rad18 ubiquitylates Pol30 during early PRR steps (Fig 3B), we began our analysis with rad6Δ cells. When compared to wild-type, rad6Δ cells are sensitive to 2.5 μg/ml cisplatin whereas hrq1Δ cells are sensitivity to 30 μg/ml cisplatin. Yeast disrupted with both HRQ1 and RAD6, exhibit sensitivity comparable to rad6Δ alone (Fig 3C). These results suggest that HRQ1 likely functions in the PRR pathway downstream of RAD6. After Rad6/Rad18 mono-ubiquitinates PCNA, PCNA becomes poly-ubiquitinated on lysine 164 during error-free PRR (Fig 3B). At the same time, PCNA can be independently sumoylated on lysine 127, which recruits the DNA helicase Srs2 (not drawn). To address whether HRQ1 functions downstream of PCNA ubiquitylation, we examined the genetic relationship between HRQ1 and PCNA ubiquitylation/sumo mutants, POL30-K164R and POL30-K127R/K164R. Disruption of HRQ1 in combination with POL30-K164R or POL30-K127R/K164R, results in cisplatin sensitivity comparable to either POL30 single mutants (Fig 3D). These results are consistent with HRQ1 functioning downstream of POL30 ubiquitylation/sumoylation during PRR. We next asked whether Hrq1 functions in the “error-free” template switching or “error-prone” translesion synthesis branches of PRR. To do this, we first examined the genetic relationship between HRQ1 and members of the “error-free” branch of PRR, RAD5-UBC13-MMS2, which polyubiquitylates PCNA (Fig 3B). Cells with both HRQ1 and RAD5 disrupted exhibit cisplatin sensitivity comparable to a RAD5 single mutant (Fig 3E). These findings are consistent with a plant study demonstrating that RAD5 functions in the same pathway as HRQ1, and contrast with a budding yeast study, where HRQ1 was found to function independently of RAD5 [44–46]. Suggesting that HRQ1 does indeed function during TS, we observe similar genetic results with another PRR member, UBC13, where hrq1Δ ubc13Δ double mutants exhibit cisplatin sensitivity to ubc13Δ (Fig 3E). Together, these results suggest that HRQ1 functions in the “error-free” branch of PRR. Next, we determined if HRQ1 also functions in the “error-prone” translesion synthesis branch of PRR by examining the genetic relationship between HRQ1 and REV1, a translesion synthesis polymerase. We observe that hrq1Δ rev1Δ double mutant cells exhibit increased cisplatin sensitivity in comparison to either a hrq1Δ or a rev1Δ single mutant (Fig 3F; 20 μg/ml cisplatin). These results suggest that Hrq1 functions primarily in the “error-free” branch of PRR.

Hrq1 function and regulation in ICL repair is distinct from intrastrand crosslink repair Loss of HRQ1 results in sensitivity to different types of DNA crosslinking agents including cisplatin and MMC. While both cisplatin and MMC cause ICLs and intrastrand crosslinks, cisplatin damage results in 90–95% intrastrand crosslinks whereas mitomycin C results in 90–95% ICLs. Therefore, by using MMC, we asked if Hrq1 has a similar function during ICL repair when ICLs are the predominant lesion. To address this, we examined hrq1Δ cells for sensitivity to MMC either alone or in combination with a rev1Δ or ubc13Δ mutant (S3 Fig). As reported in a prior study [21], hrq1Δ cells are MMC sensitive (S3A Fig). Like cisplatin induced DNA damage, the hrq1Δ rev1Δ double mutant exhibits increased MMC sensitive compared to the single mutants (S3A Fig). These results suggest that HRQ1 and REV1 are functioning in different pathways upon MMC exposure. However, in contrast to what we observed with cisplatin damage, a hrq1Δ ubc13Δ double mutant exhibits increased MMC sensitivity compared to the single hrq1Δ or ubc13Δ mutants (S3A Fig). These results suggest that Hrq1 function in ICL repair may be functionally distinct from intrastrand crosslink repair. Since we observe genetic differences in HRQ1 response to cisplatin- and MMC-induced DNA damage, we asked whether Hrq1 protein levels are similarly regulated upon MMC exposure by performing cycloheximide chase experiments (S3B Fig). Quite surprisingly, and in contrast to the cisplatin-induced response, Hrq1 protein levels are largely stable following MMC exposure (compare S3B Fig to Fig 1C). While it is possible that higher doses of MMC may result in Hrq1 degradation, together our genetic and molecular results suggest that Hrq1 function and regulation in response to ICLs is indeed distinct from intrastrand crosslinks. To validate Hrq1 role in mediating repair of intrastrand crosslinks during PRR, we performed genetic analysis using UV-C, which induces primarily 6–4 photoproducts and cyclobutane pyrimidine dimers. Suggesting a role in template switch, we find that a hrq1Δ rad5Δ double mutant has similar sensitivity to UV-C compared to rad5Δ. We observe similar results with UBC13, as a hrq1Δ ubc13Δ double mutant has similar sensitivity to UV-C as a ubc13Δ single mutant (S3C Fig). Suggesting HRQ1 functions in a different pathway to TLS, a hrq1Δ rev1Δ double mutant exhibits increased UV-C sensitive compared to the single mutants (S3D Fig). Together these results strengthen the conclusion that Hrq1 functions during intrastrand crosslink repair via PRR.

Preventing Hrq1 degradation results in increased recombination and mutations Since we find that Hrq1 functions during “error-free” PRR during intrastrand crosslink repair and is regulated by the 26S proteasome, we sought to determine if misregulation of Hrq1 protein levels alter PRR. To do this, we determined the lysine residues that catalyze Hrq1 degradation upon cisplatin exposure (Fig 4A). Hrq1 is 1077 amino acids and contains 77 lysines. To identify potential ubiquitylation sites, we analyzed Hrq1 protein using UbPred, which predicts five ubiquitylated lysines (K164, K219, K221, K366, K872). In addition, we included two lysines that are conserved between Hrq1 and its mammalian ortholog, RECQL4 (K839, K938), as well as one lysine (K366), which is both predicted to be ubiquitylated by UbPred and conserved with RECQL4 [54,55]. We mutated these seven lysine residues to arginine at the endogenous HRQ1 locus and herein refer to this mutant as Hrq1-7KR. We next determined whether mutating these Hrq1 lysine residues results in Hrq1 protein stabilization. To test this, we performed cycloheximide chases on Hrq1-3xHA and Hrq1-7KR-6xHA. As previously observed, Hrq1 protein levels are reduced upon cisplatin treatment (Figs 1C and 4B). In contrast, Hrq1-7KR protein expression remains similar in both cisplatin treated and untreated conditions (Fig 4B). It is interesting to note that the Hrq1-7KR mutant is not fully stabilized, suggesting that additional lysine residues may contribute to Hrq1 degradation independently of its DNA damage response function. These results suggest that the Hrq1-7KR mutant protein levels are misregulated in response to cisplatin. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Hrq1 protein levels are stabilized following cisplatin exposure by mutating the predicted Hrq1 ubiquitylated lysine residues to arginine. (A) Schematic of Hrq1 with lysines residues that are predicted to be ubiquitylated (K164, K219, K221, K872) or conserved between Hrq1 and RecQL4 (K366, K839, K938) is shown. The helicase domain is indicated in red (287–496 aa, InterPro). (B) Hrq1 protein levels are stabilized in Hrq1-7KR mutant. Cycloheximide chase experiments were performed in Hrq1-3xHA or Hrq1-7KR-6xHA expressing cells. Equal number of cells were collected every 30 minutes for 120 minutes. The experiment was performed in duplicate and a representative image is shown. (C) Overexpression or stabilization of Hrq1 leads to increased recombination following cisplatin treatment (35 μg/ml, 16 hours). Schematic of direct repeat recombination (DRR) assay, where recombinants are measured by formation of LEU+ recombinants (graphed). In this assay, two leu2 heteroalleles are disrupted by insertion of an EcoR1 or BsteII restriction sites, respectively, with an intervening URA3 gene. Restoration of LEU+ can occur by Rad51-dependent gene conversion (GC) measured by URA3+ LEU2+ recombinants whereas Rad51-independent single-strand annealing (SSA) is measured by URA3- LEU2+ recombinants. Nine independent colonies were measured for each experiment and the median value from two experiments (horizontal bar) were plotted (Colony count in S3 and S4 Data). ** represents p<0.01, *** represents p<0.001 (D) The average rates of GC and SSA for each condition are shown, average rates with SEM are graphed. (E) Overexpression or stabilization of Hrq1 leads to increased mutagenesis following cisplatin treatment (30 μg/ml, 16 hours). Each measurement (dots) and the median value from three experiments (horizontal bar) were plotted (Colony count in S5 Data). * represents p<0.05, ** represents p<0.01. https://doi.org/10.1371/journal.pgen.1010122.g004 We have thus far identified a function for Hrq1 in error-free PRR during intrastrand crosslink repair and found that Hrq1 targeted for degradation upon cisplatin treatment. Therefore, we hypothesized that Hrq1 may need to be degraded for completion of PRR. Since Hrq1 functions during template switching, which is a recombination-based pathway, we asked whether stabilization of Hrq1 protein levels leads to increased recombination. To test this, we utilized the Hrq1-7KR mutant strain. At the same time, we created a strain where we induce Hrq1 over-expression (Hrq1 OE) by replacing HRQ1’s endogenous promoter with a galactose-inducible/dextrose-repressible GAL1 promoter. GAL-3xHA-Hrq1 results in an approximately five-fold increase in Hrq1 expression compared to Hrq1-3xHA (S4A Fig). Therefore, we used a Hrq1-7KR strain characterized above for our studies but treated the cells in the same manner as the GAL-3xHA-Hrq1 cells to enable direct comparisons between the two strains. Using the Hrq1-7KR-6xHA mutant and the GAL-3xHA-Hrq1 strains, we performed a direct repeat recombination assay in the presence of galactose containing rich medium. We find that over-expression or stabilization of Hrq1 results in a 1.5 to 2-fold increase in total recombination compared to WT cells following cisplatin treatment (Fig 4C). The recombinant colonies in the direct repeat recombination assay can be formed by gene conversion or single-strand annealing. We then asked whether increased Hrq1 or Hrq1-7KR results in more gene conversion or single-strand annealing. We observe that both gene conversion and single-strand annealing are increased upon cisplatin exposure in the GAL-Hrq1 and Hrq1-7KR cells compared to wild-type (Fig 4D). These results suggest high levels of Hrq1 following cisplatin exposure results in increased homology-directed repair. Although recombination during template switching is considered “error-free”, aberrant template switching can result in increased DNA mutations [56,57]. Therefore, we examined whether Hrq1 over-expression or stabilization increases mutagenesis. To measure mutation rates, we performed a canavanine mutagenesis assay. This assay measures mutations in the CAN1 permease gene, which enables cell viability upon exposure to the toxic arginine analog, canavanine. We observe over two-fold increase in mutation rates in Hrq1 over-expressing or 7KR cells in the presence cisplatin in comparison to WT cells (Fig 4E). These results suggest that Hrq1 must be tightly regulated to prevent excess recombination and mutagenesis. Lastly, it is possible that preventing the degradation of Hrq1 results in accumulation of toxic DNA repair intermediates. To test this hypothesis, we examined the sensitivity of Hrq1-7KR cells upon cisplatin exposure (S4B Fig). Surprisingly, we do not observe decreased cell viability of Hrq1-7KR cells compared to WT (S4B Fig). This suggests that stabilization of Hrq1 during PRR may result in DNA repair intermediates that are ultimately resolved using an alternative pathway. Although we do not observe cisplatin sensitivity in the Hrq1-KR mutant, overexpression of the HRQ1-7KR (by using a galactose inducible promoter) results in cell lethality even in the absence of DNA damage (S4C Fig).

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