The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers.

The human SMC5/6 complex is a conserved guardian of genome stability and an emerging component of antiviral responses. These disparate functions likely require distinct mechanisms of SMC5/6 regulation. In yeast, Smc5/6 is regulated by its Nse5/6 subunits, but such regulatory subunits for human SMC5/6 are poorly defined. Here, we identify a novel SMC5/6 subunit called SIMC1 that contains SUMO interacting motifs (SIMs) and an Nse5-like domain. We isolated SIMC1 from the proteomic environment of SMC5/6 within polyomavirus large T antigen (LT)-induced subnuclear compartments. SIMC1 uses its SIMs and Nse5-like domain to localize SMC5/6 to polyomavirus replication centers (PyVRCs) at SUMO-rich PML nuclear bodies. SIMC1's Nse5-like domain binds to the putative Nse6 orthologue SLF2 to form an anti-parallel helical dimer resembling the yeast Nse5/6 structure. SIMC1-SLF2 structure-based mutagenesis defines a conserved surface region containing the N-terminus of SIMC1's helical domain that regulates SMC5/6 localization to PyVRCs. Furthermore, SLF1, which recruits SMC5/6 to DNA lesions via its BRCT and ARD motifs, binds SLF2 analogously to SIMC1 and forms a separate Nse5/6-like complex. Thus, two Nse5/6-like complexes with distinct recruitment domains control human SMC5/6 localization.

FAM111A induces nuclear dysfunction in disease and viral restriction.

Mutations in the nuclear trypsin-like serine protease FAM111A cause Kenny-Caffey syndrome (KCS2) with hypoparathyroidism and skeletal dysplasia or perinatally lethal osteocraniostenosis (OCS). In addition, FAM111A was identified as a restriction factor for certain host range mutants of the SV40 polyomavirus and VACV orthopoxvirus. However, because FAM111A function is poorly characterized, its roles in restricting viral replication and the etiology of KCS2 and OCS remain undefined. We find that FAM111A KCS2 and OCS patient mutants are hyperactive and cytotoxic, inducing apoptosis-like phenotypes such as disruption of nuclear structure and pore distribution, in a protease-dependent manner. Moreover, wild-type FAM111A activity causes similar nuclear phenotypes, including the loss of nuclear barrier function, when SV40 host range mutants attempt to replicate in restrictive cells. Interestingly, pan-caspase inhibitors do not block these FAM111A-induced phenotypes, implying it acts independently or upstream of caspases. In this regard, we identify nucleoporins and the associated GANP transcription/replication factor as FAM111A interactors and candidate targets. Overall, we reveal a potentially unifying mechanism through which deregulated FAM111A activity restricts viral replication and causes KCS2 and OCS.

Telomere length heterogeneity in ALT cells is maintained by PML-dependent localization of the BTR complex to telomeres.

Telomeres consist of TTAGGG repeats bound by protein complexes that serve to protect the natural end of linear chromosomes. Most cells maintain telomere repeat lengths by using the enzyme telomerase, although there are some cancer cells that use a telomerase-independent mechanism of telomere extension, termed alternative lengthening of telomeres (ALT). Cells that use ALT are characterized, in part, by the presence of specialized PML nuclear bodies called ALT-associated PML bodies (APBs). APBs localize to and cluster telomeric ends together with telomeric and DNA damage factors, which led to the proposal that these bodies act as a platform on which ALT can occur. However, the necessity of APBs and their function in the ALT pathway has remained unclear. Here, we used CRISPR/Cas9 to delete PML and APB components from ALT-positive cells to cleanly define the function of APBs in ALT. We found that PML is required for the ALT mechanism, and that this necessity stems from APBs' role in localizing the BLM-TOP3A-RMI (BTR) complex to ALT telomere ends. Strikingly, recruitment of the BTR complex to telomeres in a PML-independent manner bypasses the need for PML in the ALT pathway, suggesting that BTR localization to telomeres is sufficient to sustain ALT activity.

Recruitment, loading, and activation of the Smc5-Smc6 SUMO ligase.

Duplication of the genome poses one of the most significant threats to genetic integrity, cellular fitness, and organismal health. Therefore, numerous mechanisms have evolved that maintain replication fork stability in the face of DNA damage and allow faithful genome duplication. The fission yeast BRCT-domain-containing protein Brc1, and its budding yeast orthologue Rtt107, has emerged as a "hub" factor that integrates multiple replication fork protection mechanisms. Notably, the cofactors and pathways through which Brc1, Rtt107, and their human orthologue (PTIP) act have appeared largely distinct. This either represents true evolutionary functional divergence, or perhaps an incomplete genetic and biochemical analysis of each protein. In this regard, we recently showed that like Rtt107, Brc1 supports key functions of the Smc5-Smc6 complex, including its recruitment into DNA repair foci, chromatin association, and SUMO ligase activity. Furthermore, fission yeast cells lacking the Nse5-Nse6 genome stability factor were found to exhibit defects in Smc5-Smc6 function, similar to but more severe than those in cells lacking Brc1. Here, we place these findings in context with the known functions of Brc1, Rtt107, and Smc5-Smc6, present data suggesting a role for acetylation in Smc5-Smc6 chromatin loading, and discuss wider implications for genome stability.

Brc1 Promotes the Focal Accumulation and SUMO Ligase Activity of Smc5-Smc6 during Replication Stress.

As genetic instability drives disease or loss of cell fitness, cellular safeguards have evolved to protect the genome, especially during sensitive cell cycle phases, such as DNA replication. Fission yeast Brc1 has emerged as a key factor in promoting cell survival when replication forks are stalled or collapsed. Brc1 is a multi-BRCT protein that is structurally related to the budding yeast Rtt107 and human PTIP DNA damage response factors, but functional similarities appear limited. Brc1 is a dosage suppressor of a mutation in the essential Smc5-Smc6 genome stability complex and is thought to act in a bypass pathway. In this study, we reveal an unexpectedly intimate connection between Brc1 and Smc5-Smc6 function. Brc1 is required for the accumulation of the Smc5-Smc6 genome stability complex in foci during replication stress and for activation of the intrinsic SUMO ligase activity of the complex by collapsed replication forks. Moreover, we show that the chromatin association and SUMO ligase activity of Smc5-Smc6 require the Nse5-Nse6 heterodimer, explaining how this nonessential cofactor critically supports the DNA repair roles of Smc5-Smc6. We also found that Brc1 interacts with Nse5-Nse6, as well as gamma-H2A, so it can tether Smc5-Smc6 at replicative DNA lesions to promote survival.

SUMO-targeted ubiquitin ligase activity can either suppress or promote genome instability, depending on the nature of the DNA lesion.

The posttranslational modifiers SUMO and ubiquitin critically regulate the DNA damage response (DDR). Important crosstalk between these modifiers at DNA lesions is mediated by the SUMO-targeted ubiquitin ligase (STUbL), which ubiquitinates SUMO chains to generate SUMO-ubiquitin hybrids. These SUMO-ubiquitin hybrids attract DDR proteins able to bind both modifiers, and/or are degraded at the proteasome. Despite these insights, specific roles for SUMO chains and STUbL in the DDR remain poorly defined. Notably, fission yeast defective in SUMO chain formation exhibit near wild-type resistance to genotoxins and moreover, have a greatly reduced dependency on STUbL activity for DNA repair. Based on these and other data, we propose that a critical role of STUbL is to antagonize DDR-inhibitory SUMO chain formation at DNA lesions. In this regard, we identify a SUMO-binding Swi2/Snf2 translocase called Rrp2 (ScUls1) as a mediator of the DDR defects in STUbL mutant cells. Therefore, in support of our proposal, SUMO chains attract activities that can antagonize STUbL and other DNA repair factors. Finally, we find that Taz1TRF1/TRF2-deficiency triggers extensive telomeric poly-SUMOylation. In this setting STUbL, together with its cofactor Cdc48p97, actually promotes genomic instability caused by the aberrant processing of taz1Δ telomeres by DNA repair factors. In summary, depending on the nature of the initiating DNA lesion, STUbL activity can either be beneficial or harmful.

Large-Scale Purification of Small Ubiquitin-Like Modifier (SUMO)-Modified Proteins from Schizosaccharomyces pombe.

Covalent protein modification by sumoylation (i.e., addition of small ubiquitin-like modifiers [SUMOs]) regulates a broad spectrum of critical functions in eukaryotic cells; however, usually ≤1% of a given protein is modified as a result of the highly dynamic nature of sumoylation. As such, capturing and identifying sumoylated proteins are both important in biological studies and very challenging tasks. Here we report a tailored purification protocol that includes rapid and complete cell disruption, coupled to highly stringent isolation of sumoylated proteins. Proteins purified using this protocol are compatible with common downstream applications such as western and mass spectrometry analyses. This protocol will work equally well to study other key covalent modifiers such as ubiquitin and Ned8.

Improved Tandem Affinity Purification Tag and Methods for Isolation of Proteins and Protein Complexes from Schizosaccharomyces pombe.

The tandem affinity purification (TAP) method uses an epitope that contains two different affinity purification tags separated by a site-specific protease site to isolate a protein rapidly and easily. Proteins purified via the TAP tag are eluted under mild conditions, allowing them to be used for structural and biochemical analyses. The original TAP tag contains a calmodulin-binding peptide and the IgG-binding domain from protein A separated by a tobacco etch virus (TEV) protease cleavage site. After capturing the Protein A epitope on an IgG resin, bound proteins are released by incubation with the TEV protease and then isolated on a calmodulin matrix in the presence of calcium; elution from this resin is achieved by chelating calcium with EGTA. However, because the robustness of the calmodulin-binding step in this procedure is highly variable, we replaced the calmodulin-binding peptide with three copies of the FLAG epitope, (3× FLAG)-TEV-Protein A, which can be isolated using an anti-FLAG resin. Elution from this matrix is achieved in the presence of an excess of a 3× FLAG peptide. In addition to allowing proteins to be released under mild conditions, elution by the 3× FLAG peptide adds an extra layer of specificity to the TAP procedure, because it liberates only FLAG-tagged proteins.

Stability of apomorphine in solutions containing selected antioxidant agents.

Apomorphine in solution undergoes rapid autoxidation, producing greenish colored solutions, making it difficult to formulate as a stable pharmaceutical solution. To identify the optimum antioxidant agent/combination for apomorphine solution, a high performance liquid chromatography assay was used to study the stability of 50 µg/mL apomorphine HCI in 0.1% L-ascorbic acid (AA), 0.1% sodium metabisulfite (SMB), 0.1% EDTA, and in selected combinations at 25°C, 32°C, and 37°C over a period of 14 days. The stability of apomorphine HCl (10 mg/mL) in 0.1% AA solution and in 0.1% EDTA solution at 25°C and 37°C was also evaluated. Apomorphine HCI solution (50 µg/mL) in 0.1% AA plus 0.1% SMB solution retained 99.7% (at 25°C) and 95.9% (at 37°C) of the initial concentration, as 0.1% AA plus SMB solution minimized the reactive oxygen content in solution which, in turn, reduced the oxidation rate of apomorphine HCl, and there was no green coloration perceptible. Conversely, apomorphine HCl solution (50 µg/mL) in 0.1% SMB solution was unstable as only 0.53% (at 25°C) and 0.06% (at 37°C) of the initial concentration was retained after 14 days. All 10 mg/mL apomorphine HCl samples were stable in both studies. The initial concentration of apomorphine HCl solution markedly affected its rate of oxidation and discoloration. The addition of 0.1% AA to a current formulation of apomorphine HCl injection (Apomine®), which contains SMB as an antioxidant, was recommended as providing the most stable solution.

Functional Crosstalk between the PP2A and SUMO Pathways Revealed by Analysis of STUbL Suppressor, razor 1-1.

Posttranslational modifications (PTMs) provide dynamic regulation of the cellular proteome, which is critical for both normal cell growth and for orchestrating rapid responses to environmental stresses, e.g. genotoxins. Key PTMs include ubiquitin, the Small Ubiquitin-like MOdifier SUMO, and phosphorylation. Recently, SUMO-targeted ubiquitin ligases (STUbLs) were found to integrate signaling through the SUMO and ubiquitin pathways. In general, STUbLs are recruited to target proteins decorated with poly-SUMO chains to ubiquitinate them and drive either their extraction from protein complexes, and/or their degradation at the proteasome. In fission yeast, reducing or preventing the formation of SUMO chains can circumvent the essential and DNA damage response functions of STUbL. This result indicates that whilst some STUbL "targets" have been identified, the crucial function of STUbL is to antagonize SUMO chain formation. Herein, by screening for additional STUbL suppressors, we reveal crosstalk between the serine/threonine phosphatase PP2A-Pab1B55 and the SUMO pathway. A hypomorphic Pab1B55 mutant not only suppresses STUbL dysfunction, but also mitigates the phenotypes associated with deletion of the SUMO protease Ulp2, or mutation of the STUbL cofactor Rad60. Together, our results reveal a novel role for PP2A-Pab1B55 in modulating SUMO pathway output, acting in parallel to known critical regulators of SUMOylation homeostasis. Given the broad evolutionary functional conservation of the PP2A and SUMO pathways, our results could be relevant to the ongoing attempts to therapeutically target these factors.

Stability studies of lincomycin hydrochloride in aqueous solution and intravenous infusion fluids.

PURPOSE: The purpose of this study was to evaluate the chemical stability of Lincocin(®) (lincomycin hydrochloride) in commonly used intravenous fluids at room temperature (25°C), at accelerated-degradation temperatures and in selected buffer solutions. MATERIALS AND METHODS: The stability of Lincocin(®) injection (containing lincomycin 600 mg/2 mL as the hydrochloride) stored at 25°C±0.1°C in sodium lactate (Hartmann's), 0.9% sodium chloride, 5% glucose, and 10% glucose solutions was investigated over 31 days. Forced degradation of Lincocin(®) in hydrochloric acid, sodium hydroxide, and hydrogen peroxide was performed at 60°C. The effect of pH on the degradation rate of lincomycin hydrochloride stored at 80°C was determined. RESULTS: Lincomycin hydrochloride w as found to maintain its shelf life at 25°C in sodium lactate (Hartmann's) solution, 0.9% sodium chloride solution, 5% glucose solution, and 10% glucose solution, with less than 5% lincomycin degradation occurring in all intravenous solutions over a 31-day period. Lincomycin hydrochloride showed less rapid degradation at 60°C in acid than in basic solution, but degraded rapidly in hydrogen peroxide. At all pH values tested, lincomycin followed first-order kinetics. It had the greatest stability near pH 4 when stored at 80°C (calculated shelf life of 4.59 days), and was least stable at pH 2 (calculated shelf life of 0.38 days). CONCLUSION: Lincocin(®) injection was chemically found to have a shelf life of at least 31 days at 25°C when added to sodium lactate (Hartmann's) solution, 0.9% sodium chloride solution, 5% glucose solution, and 10% glucose solution. Solutions prepared at approximately pH 4 are likely to have optimum stability.

Cooperativity of the SUMO and Ubiquitin Pathways in Genome Stability.

Covalent attachment of ubiquitin (Ub) or SUMO to DNA repair proteins plays critical roles in maintaining genome stability. These structurally related polypeptides can be viewed as distinct road signs, with each being read by specific protein interaction motifs. Therefore, via their interactions with selective readers in the proteome, ubiquitin and SUMO can elicit distinct cellular responses, such as directing DNA lesions into different repair pathways. On the other hand, through the action of the SUMO-targeted ubiquitin ligase (STUbL) family proteins, ubiquitin and SUMO can cooperate in the form of a hybrid signal. These mixed SUMO-ubiquitin chains recruit "effector" proteins such as the AAA⁺ ATPase Cdc48/p97-Ufd1-Npl4 complex that contain both ubiquitin and SUMO interaction motifs. This review will summarize recent key findings on collaborative and distinct roles that ubiquitin and SUMO play in orchestrating DNA damage responses.

High Confidence Fission Yeast SUMO Conjugates Identified by Tandem Denaturing Affinity Purification.

Covalent attachment of the small ubiquitin-like modifier (SUMO) to key targets in the proteome critically regulates the evolutionarily conserved processes of cell cycle control, transcription, DNA replication and maintenance of genome stability. The proteome-wide identification of SUMO conjugates in budding yeast has been invaluable in helping to define roles of SUMO in these processes. Like budding yeast, fission yeast is an important and popular model organism; however, the fission yeast Schizosaccharomyces pombe community currently lacks proteome-wide knowledge of SUMO pathway targets. To begin to address this deficiency, we adapted and used a highly stringent Tandem Denaturing Affinity Purification (TDAP) method, coupled with mass spectrometry, to identify fission yeast SUMO conjugates. Comparison of our data with that compiled in budding yeast reveals conservation of SUMO target enrichment in nuclear and chromatin-associated processes. Moreover, the SUMO "cloud" phenomenon, whereby multiple components of a single protein complex are SUMOylated, is also conserved. Overall, SUMO TDAP provides both a key resource of high confidence SUMO-modified target proteins in fission yeast, and a robust method for future analyses of SUMO function.

Pli1(PIAS1) SUMO ligase protected by the nuclear pore-associated SUMO protease Ulp1SENP1/2.

Covalent modification of the proteome by SUMO is critical for genetic stability and cell growth. Equally crucial to these processes is the removal of SUMO from its targets by the Ulp1 (HuSENP1/2) family of SUMO proteases. Ulp1 activity is normally spatially restricted, because it is localized to the nuclear periphery via interactions with the nuclear pore. Delocalization of Ulp1 causes DNA damage and cell cycle defects, phenotypes thought to be caused by inappropriate desumoylation of nucleoplasmic targets that are normally spatially protected from Ulp1. Here, we define a novel consequence of Ulp1 deregulation, with a major impact on SUMO pathway function. In fission yeast lacking Nup132 (Sc/HuNUP133), Ulp1 is delocalized and can no longer antagonize sumoylation of the PIAS family SUMO E3 ligase, Pli1. Consequently, SUMO chain-modified Pli1 is targeted for proteasomal degradation by the concerted action of a SUMO-targeted ubiquitin ligase (STUbL) and Cdc48-Ufd1-Npl4. Pli1 degradation causes the profound SUMO pathway defects and associated centromere dysfunction in cells lacking Nup132. Thus, perhaps counterintuitively, Ulp1-mediated desumoylation can promote SUMO modification by stabilizing a SUMO E3 ligase.

A novel histone deacetylase complex in the control of transcription and genome stability.

The acetylation state of histones, controlled by histone acetyltransferases (HATs) and deacetylases (HDACs), profoundly affects DNA transcription and repair by modulating chromatin accessibility to the cellular machinery. The Schizosaccharomyces pombe HDAC Clr6 (human HDAC1) binds to different sets of proteins that define functionally distinct complexes: I, I', and II. Here, we determine the composition, architecture, and functions of a new Clr6 HDAC complex, I'', delineated by the novel proteins Nts1, Mug165, and Png3. Deletion of nts1 causes increased sensitivity to genotoxins and deregulated expression of Tf2 elements, long noncoding RNA, and subtelomeric and stress-related genes. Similar, but more pervasive, phenotypes are observed upon Clr6 inactivation, supporting the designation of complex I'' as a mediator of a key subset of Clr6 functions. We also reveal that with the exception of Tf2 elements, the genome-wide loading sites and loci regulated by Clr6 I″ do not correlate. Instead, Nts1 loads at genes that are expressed in midmeiosis, following oxidative stress, or are periodically expressed. Collective data suggest that Clr6 I'' has (i) indirect effects on gene expression, conceivably by mediating higher-order chromatin organization of subtelomeres and Tf2 elements, and (ii) direct effects on the transcription of specific genes in response to certain cellular or environmental stimuli.

RNF4 interacts with both SUMO and nucleosomes to promote the DNA damage response.

The post-translational modification of DNA repair and checkpoint proteins by ubiquitin and small ubiquitin-like modifier (SUMO) critically orchestrates the DNA damage response (DDR). The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin, through its selective recognition and ubiquitination of SUMO-modified proteins. Here, we define a key new determinant for target discrimination by RNF4, in addition to interaction with SUMO. We identify a nucleosome-targeting motif within the RNF4 RING domain that can bind DNA and thereby enables RNF4 to selectively ubiquitinate nucleosomal histones. Furthermore, RNF4 nucleosome-targeting is crucially required for the repair of TRF2-depleted dysfunctional telomeres by 53BP1-mediated non-homologous end joining.

A new versatile system for rapid control of gene expression in the fission yeast Schizosaccharomyces pombe.

The ability to regulate the expression of a gene greatly aids the process of uncovering its functions. The fission yeast Schizosaccharomyces pombe has so far lacked a system for rapidly controlling the expression of chromosomal genes, hindering its full potential as a model organism. Although the widely used nmt1 promoter displays a wide dynamic range of activity, it takes > 14-15 h to derepress. The urg1 promoter also shows a large dynamic range and can be induced quickly (< 2 h), but its implementation requires laborious strain construction and it cannot be used to study meiosis. To overcome these limitations, we constructed a tetracycline-regulated system for inducible expression of chromosomal genes in fission yeast, which is easily established and implemented. In this system the promoter of a gene is replaced by simple one-step substitution techniques with a tetracycline-regulated promoter cassette (tetO(7) -TATA(CYC1) ) in cells where TetR/TetR'-based transcription activators/repressors are also produced. Using top1 and nse6 as reporter genes, we show that Top1 and Nse6 appear after just 30 min of activating tetO(7) -TATA(CYC1) and plateau after -4-6 h. The amount of synthesised protein is comparable to that produced from the attenuated nmt1 promoter P(nmt8) , which should be closer to wild-type levels for most genes than those generated from excessively strong promoters and can be controlled by changing the concentration of the effector antibiotic. This system also works efficiently during meiosis, thus making it a useful addition to the toolkit of the fission yeast community.

Meiotic DNA joint molecule resolution depends on Nse5-Nse6 of the Smc5-Smc6 holocomplex.

Faithful chromosome segregation in meiosis is crucial to form viable, healthy offspring and in most species, it requires programmed recombination between homologous chromosomes. In fission yeast, meiotic recombination is initiated by Rec12 (Spo11 homolog) and generates single Holliday junction (HJ) intermediates, which are resolved by the Mus81-Eme1 endonuclease to generate crossovers and thereby allow proper chromosome segregation. Although Mus81 contains the active site for HJ resolution, the regulation of Mus81-Eme1 is unclear. In cells lacking Nse5-Nse6 of the Smc5-Smc6 genome stability complex, we observe persistent meiotic recombination intermediates (DNA joint molecules) resembling HJs that accumulate in mus81Δ cells. Elimination of Rec12 nearly completely rescues the meiotic defects of nse6Δ and mus81Δ single mutants and partially rescues nse6Δ mus81Δ double mutants, indicating that these factors act after DNA double-strand break formation. Likewise, expression of the bacterial HJ resolvase RusA partially rescues the defects of nse6Δ, mus81Δ and nse6Δ mus81Δ mitotic cells, as well as the meiotic defects of nse6Δ and mus81Δ cells. Partial rescue likely reflects the accumulation of structures other than HJs, such as hemicatenanes, and an additional role for Nse5-Nse6 most prominent during mitotic growth. Our results indicate a regulatory role for the Smc5-Smc6 complex in HJ resolution via Mus81-Eme1.

Dual recruitment of Cdc48 (p97)-Ufd1-Npl4 ubiquitin-selective segregase by small ubiquitin-like modifier protein (SUMO) and ubiquitin in SUMO-targeted ubiquitin ligase-mediated genome stability functions.

Protein modification by SUMO and ubiquitin critically impacts genome stability via effectors that "read" their signals using SUMO interaction motifs or ubiquitin binding domains, respectively. A novel mixed SUMO and ubiquitin signal is generated by the SUMO-targeted ubiquitin ligase (STUbL), which ubiquitylates SUMO conjugates. Herein, we determine that the "ubiquitin-selective" segregase Cdc48-Ufd1-Npl4 also binds SUMO via a SUMO interaction motif in Ufd1 and can thus act as a selective receptor for STUbL targets. Indeed, we define key cooperative DNA repair functions for Cdc48-Ufd1-Npl4 and STUbL, thereby revealing a new signaling mechanism involving dual recruitment by SUMO and ubiquitin for Cdc48-Ufd1-Npl4 functions in maintaining genome stability.

The RecQ4 orthologue Hrq1 is critical for DNA interstrand cross-link repair and genome stability in fission yeast.

Of the five human RecQ family helicases, RecQ4, BLM, and WRN suppress distinct genome instability-linked diseases with severe phenotypes, often with indeterminate etiologies. Here, we functionally define Hrq1, a novel orthologue of RecQ4 from fission yeast. Biochemical analysis of Hrq1 reveals a DEAH box- and ATP-dependent 3'-5' helicase activity on various DNA substrates, including bubbles but not blunt duplexes, characteristic of the RecQ family. Cells lacking Hrq1 suffer spontaneous genomic instability and, consequently, require homologous recombination repair and the DNA damage checkpoint for viability. Hrq1 supports the nucleotide excision repair of DNA damage caused by the chemotherapeutic agent cisplatin and, in certain genetic contexts, UV light. Genetic epistasis analyses reveal that Hrq1 acts parallel to the PCNA/Ubc13/Mms2-dependent postreplication repair (PRR) pathway. Thus, in hrq1Δ cells, lesions are channeled through the PRR pathway, yielding hyper-recombinant and mutator phenotypes; analogous defects may underlie the genetic instability and diseases associated with RecQ4 dysfunction.