Loss of Dna2 fidelity results in decreased Exo1-mediated resection at DNA double-strand breaks.

A DNA double-strand break (DSB) is one of the most dangerous types of DNA damage that is repaired largely by homologous recombination or nonhomologous end-joining (NHEJ). The interplay of repair factors at the break directs which pathway is used, and a subset of these factors also function in more mutagenic alternative (alt) repair pathways. Resection is a key event in repair pathway choice and extensive resection, which is a hallmark of homologous recombination, and it is mediated by two nucleases, Exo1 and Dna2. We observed differences in resection and repair outcomes in cells harboring nuclease-dead dna2-1 compared with dna2Δ pif1-m2 that could be attributed to the level of Exo1 recovered at DSBs. Cells harboring dna2-1 showed reduced Exo1 localization, increased NHEJ, and a greater resection defect compared with cells where DNA2 was deleted. Both the resection defect and the increased rate of NHEJ in dna2-1 mutants were reversed upon deletion of KU70 or ectopic expression of Exo1. By contrast, when DNA2 was deleted, Exo1 and Ku70 recovery levels did not change; however, Nej1 increased as did the frequency of alt-end joining/microhomology-mediated end-joining repair. Our findings demonstrate that decreased Exo1 at DSBs contributed to the resection defect in cells expressing inactive Dna2 and highlight the complexity of understanding how functionally redundant factors are regulated in vivo to promote genome stability.

SIR telomere silencing depends on nuclear envelope lipids and modulates sensitivity to a lysolipid.

The nuclear envelope (NE) is important in maintaining genome organization. The role of lipids in communication between the NE and telomere regulation was investigated, including how changes in lipid composition impact gene expression and overall nuclear architecture. Yeast was treated with the non-metabolizable lysophosphatidylcholine analog edelfosine, known to accumulate at the perinuclear ER. Edelfosine induced NE deformation and disrupted telomere clustering but not anchoring. Additionally, the association of Sir4 at telomeres decreased. RNA-seq analysis showed altered expression of Sir-dependent genes located at sub-telomeric (0-10 kb) regions, consistent with Sir4 dispersion. Transcriptomic analysis revealed that two lipid metabolic circuits were activated in response to edelfosine, one mediated by the membrane sensing transcription factors, Spt23/Mga2, and the other by a transcriptional repressor, Opi1. Activation of these transcriptional programs resulted in higher levels of unsaturated fatty acids and the formation of nuclear lipid droplets. Interestingly, cells lacking Sir proteins displayed resistance to unsaturated-fatty acids and edelfosine, and this phenotype was connected to Rap1.

Changes in DNA double-strand break repair during aging correlate with an increase in genomic mutations.

A double -strand break (DSB) is one of the most deleterious forms of DNA damage. In eukaryotic cells, two main repair pathways have evolved to repair DSBs, homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is the predominant pathway of repair in the unicellular eukaryotic organism, S. cerevisiae. However, during replicative aging the relative use of HR and NHEJ shifts in favor of end-joining repair. By monitoring repair events in the HO-DSB system, we find that early in replicative aging there is a decrease in the association of long-range resection factors, Dna2-Sgs1 and Exo1 at the break site and a decrease in DNA resection. Subsequently, as aging progressed, the recovery of Ku70 at DSBs decreased and the break site associated with the nuclear pore complex at the nuclear periphery, which is the location where DSB repair occurs through alternative pathways that are more mutagenic. End-bridging remained intact as HR and NHEJ declined, but eventually it too became disrupted in cells at advanced replicative age. In all, our work provides insight into the molecular changes in DSB repair pathway during replicative aging. HR first declined, resulting in a transient increase in the NHEJ. However, with increased cellular divisions, Ku70 recovery at DSBs and NHEJ subsequently declined. In wild type cells of advanced replicative age, there was a high frequency of repair products with genomic deletions and microhomologies at the break junction, events not observed in young cells which repaired primarily by HR.

Multifunctional properties of Nej1XLF C-terminus promote end-joining and impact DNA double-strand break repair pathway choice.

A DNA double strand break (DSB) is primarily repaired by one of two canonical pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ requires no or minimal end processing for ligation, whereas HR requires 5' end resection followed by a search for homology. The main event that determines the mode of repair is the initiation of 5' resection because if resection starts, then NHEJ cannot occur. Nej1 is a canonical NHEJ factor that functions at the cross-roads of repair pathway choice and prior to its function in stimulating Dnl4 ligase. Nej1 competes with Dna2, inhibiting its recruitment to DSBs and thereby inhibiting resection. The highly conserved C-terminal region (CTR) of Nej1 (330-338) is important for two events that drive NHEJ as it stimulates ligation and inhibits resection, but it is dispensable for end-bridging. By combining nej1 point mutants with nuclease-dead dna2-1, we find that Nej1-F335 is essential for end-joining whereas V338 promotes NHEJ indirectly by inhibiting Dna2-mediated resection.

Nej1 interacts with Sae2 at DNA double-stranded breaks to inhibit DNA resection.

The two major pathways of DNA double-strand break repair, nonhomologous end-joining and homologous recombination, are highly conserved from yeast to mammals. The regulation of 5'-DNA resection controls repair pathway choice and influences repair outcomes. Nej1 was first identified as a canonical NHEJ factor involved in stimulating the ligation of broken DNA ends, and more recently, it was shown to participate in DNA end-bridging and in the inhibition of 5'-resection mediated by the nuclease/helicase complex Dna2-Sgs1. Here, we show that Nej1 interacts with Sae2 to impact DSB repair in three ways. First, we show that Nej1 inhibits interaction of Sae2 with the Mre11-Rad50-Xrs2 complex and Sae2 localization to DSBs. Second, we found that Nej1 inhibits Sae2-dependent recruitment of Dna2 independently of Sgs1. Third, we determined that NEJ1 and SAE2 showed an epistatic relationship for end-bridging, an event that restrains broken DNA ends and reduces the frequency of genomic deletions from developing at the break site. Finally, we demonstrate that deletion of NEJ1 suppressed the synthetic lethality of sae2Δ sgs1Δ mutants, and that triple mutant viability was dependent on Dna2 nuclease activity. Taken together, these findings provide mechanistic insight to how Nej1 functionality inhibits the initiation of DNA resection, a role that is distinct from its involvement in end-joining repair at DSBs.

Smc5/6 in the rDNA modulates lifespan independently of Fob1​.

The ribosomal DNA (rDNA) in Saccharomyces cerevisiae is in one tandem repeat array on Chromosome XII. Two regions within each repetitive element, called intergenic spacer 1 (IGS1) and IGS2, are important for organizing the rDNA within the nucleolus. The Smc5/6 complex localizes to IGS1 and IGS2. We show that Smc5/6 has a function in the rDNA beyond its role in homologous recombination (HR) at the replication fork barrier (RFB) located in IGS1. Fob1 is required for optimal binding of Smc5/6 at IGS1 whereas the canonical silencing factor Sir2 is required for its optimal binding at IGS2, independently of Fob1. Through interdependent interactions, Smc5/6 stabilizes Sir2 and Cohibin at both IGS and its recovery at IGS2 is important for nucleolar compaction and transcriptional silencing, which in turn supports rDNA stability and lifespan.

A Role for the Mre11-Rad50-Xrs2 Complex in Gene Expression and Chromosome Organization.

Mre11-Rad50-Xrs2 (MRX) is a highly conserved complex with key roles in various aspects of DNA repair. Here, we report a new function for MRX in limiting transcription in budding yeast. We show that MRX interacts physically and colocalizes on chromatin with the transcriptional co-regulator Mediator. MRX restricts transcription of coding and noncoding DNA by a mechanism that does not require the nuclease activity of Mre11. MRX is required to tether transcriptionally active loci to the nuclear pore complex (NPC), and it also promotes large-scale gene-NPC interactions. Moreover, MRX-mediated chromatin anchoring to the NPC contributes to chromosome folding and helps to control gene expression. Together, these findings indicate that MRX has a role in transcription and chromosome organization that is distinct from its known function in DNA repair.

A high-throughput alpha particle irradiation system for monitoring DNA damage repair, genome instability and screening in human cell and yeast model systems.

Ionizing radiation (IR) is environmentally prevalent and, depending on dose and linear energy transfer (LET), can elicit serious health effects by damaging DNA. Relative to low LET photon radiation (X-rays, gamma rays), higher LET particle radiation produces more disease causing, complex DNA damage that is substantially more challenging to resolve quickly or accurately. Despite the majority of human lifetime IR exposure involving long-term, repetitive, low doses of high LET alpha particles (e.g. radon gas inhalation), technological limitations to deliver alpha particles in the laboratory conveniently, repeatedly, over a prolonged period, in low doses and in an affordable, high-throughput manner have constrained DNA damage and repair research on this topic. To resolve this, we developed an inexpensive, high capacity, 96-well plate-compatible alpha particle irradiator capable of delivering adjustable, low mGy/s particle radiation doses in multiple model systems and on the benchtop of a standard laboratory. The system enables monitoring alpha particle effects on DNA damage repair and signalling, genome stability pathways, oxidative stress, cell cycle phase distribution, cell viability and clonogenic survival using numerous microscopy-based and physical techniques. Most importantly, this method is foundational for high-throughput genetic screening and small molecule testing in mammalian and yeast cells.

SUNny Ways: The Role of the SUN-Domain Protein Mps3 Bridging Yeast Nuclear Organization and Lipid Homeostasis.

Mps3 is a SUN (Sad1-UNC-84) domain-containing protein that is located in the inner nuclear membrane (INM). Genetic screens with multiple Mps3 mutants have suggested that distinct regions of Mps3 function in relative isolation and underscore the broad involvement of Mps3 in multiple pathways including mitotic spindle formation, telomere maintenance, and lipid metabolism. These pathways have largely been characterized in isolation, without a holistic consideration for how key regulatory events within one pathway might impinge on other aspects of biology at the nuclear membrane. Mps3 is uniquely positioned to function in these multiple pathways as its N- terminus is in the nucleoplasm, where it is important for telomere anchoring at the nuclear periphery, and its C-terminus is in the lumen, where it has links with lipid metabolic processes. Emerging work suggests that the role of Mps3 in nuclear organization and lipid homeostasis are not independent, but more connected. For example, a failure in regulating Mps3 levels through the cell cycle leads to nuclear morphological abnormalities and loss of viability, suggesting a link between the N-terminal domain of Mps3 and nuclear envelope homeostasis. We will highlight work suggesting that Mps3 is pivotal factor in communicating events between the nucleus and the lipid bilayer.

Modeling cancer genomic data in yeast reveals selection against ATM function during tumorigenesis.

The DNA damage response (DDR) comprises multiple functions that collectively preserve genomic integrity and suppress tumorigenesis. The Mre11 complex and ATM govern a major axis of the DDR and several lines of evidence implicate that axis in tumor suppression. Components of the Mre11 complex are mutated in approximately five percent of human cancers. Inherited mutations of complex members cause severe chromosome instability syndromes, such as Nijmegen Breakage Syndrome, which is associated with strong predisposition to malignancy. And in mice, Mre11 complex mutations are markedly more susceptible to oncogene- induced carcinogenesis. The complex is integral to all modes of DNA double strand break (DSB) repair and is required for the activation of ATM to effect DNA damage signaling. To understand which functions of the Mre11 complex are important for tumor suppression, we undertook mining of cancer genomic data from the clinical sequencing program at Memorial Sloan Kettering Cancer Center, which includes the Mre11 complex among the 468 genes assessed. Twenty five mutations in MRE11 and RAD50 were modeled in S. cerevisiae and in vitro. The mutations were chosen based on recurrence and conservation between human and yeast. We found that a significant fraction of tumor-borne RAD50 and MRE11 mutations exhibited separation of function phenotypes wherein Tel1/ATM activation was severely impaired while DNA repair functions were mildly or not affected. At the molecular level, the gene products of RAD50 mutations exhibited defects in ATP binding and hydrolysis. The data reflect the importance of Rad50 ATPase activity for Tel1/ATM activation and suggest that inactivation of ATM signaling confers an advantage to burgeoning tumor cells.

Nej1 Interacts with Mre11 to Regulate Tethering and Dna2 Binding at DNA Double-Strand Breaks.

Non-homologous end joining (NHEJ) and homologous recombination (HR) are the two major pathways of DNA double-strand break (DSB) repair and both are highly conserved from yeast to mammals. Nej1 has a role in DNA end-tethering at a DSB, and the Mre11/Rad50/Xrs2 (MRX) complex is important for its recruitment to the break. Nej1 and Dna2-Sgs1 interact with the C-terminal end of Mre11, which also includes the region where Rad50 binds. By characterizing the functionality of Nej1 in two rad50 mutants, which alter the structural features of MRX, we demonstrate that Nej1 inhibits the binding of Dna2 to Mre11 and Sgs1. Nej1 interactions with Mre11 promote tethering and inhibit hyper-resection, and when these events are compromised, large deletions develop at a DSB. The work indicates that Nej1 provides a layer of regulation to repair pathway choice and is consistent with its role in NHEJ.

The non-homologous end-joining factor Nej1 inhibits resection mediated by Dna2-Sgs1 nuclease-helicase at DNA double strand breaks.

Double strand breaks (DSBs) represent highly deleterious DNA damage and need to be accurately repaired. Homology-directed repair and non-homologous end joining (NHEJ) are the two major DSB repair pathways that are highly conserved from yeast to mammals. The choice between these pathways is largely based on 5' to 3' DNA resection, and NHEJ proceeds only if resection has not been initiated. In yeast, yKu70/80 rapidly localizes to the break, protecting DNA ends from nuclease accessibility, and recruits additional NHEJ factors, including Nej1 and Lif1. Cells harboring the nej1-V338A mutant exhibit NHEJ-mediated repair deficiencies and hyper-resection 0.15 kb from the DSB that was dependent on the nuclease activity of Dna2-Sgs1. The integrity of Nej1 is also important for inhibiting long-range resection, 4.8 kb from the break, and for preventing the formation of large genomic deletions at sizes >700 bp around the break. Nej1V338A localized to a DSB similarly to WT Nej1, indicating that the Nej1-Lif1 interaction becomes critical for blocking hyper-resection mainly after their recruitment to the DSB. This work highlights that Nej1 inhibits 5' DNA hyper-resection mediated by Dna2-Sgs1, a function distinct from its previously reported role in supporting Dnl4 ligase activity, and has implications for repair pathway choice and resection regulation upon DSB formation.

A decade of understanding spatio-temporal regulation of DNA repair by the nuclear architecture.

The nucleus is a hub for gene expression and is a highly organized entity. The nucleoplasm is heterogeneous, owing to the preferential localization of specific metabolic factors, which lead to the definition of nuclear compartments or bodies. The genome is organized into chromosome territories, as well as heterochromatin and euchromatin domains. Recent observations have indicated that nuclear organization is important for maintaining genomic stability. For example, nuclear organization has been implicated in stabilizing damaged DNA, repair-pathway choice, and in preventing chromosomal rearrangements. Over the past decade, several studies have revealed that dynamic changes in the nuclear architecture are important during double-strand break repair. Stemming from work in yeast, relocation of a damaged site prior to repair appears to be at least partially conserved in multicellular eukaryotes. In this review, we will discuss genome and nucleoplasm architecture, particularly the importance of the nuclear periphery in genome stability. We will also discuss how the site of relocation regulates repair-pathway choice.

Smc5/6 Is a Telomere-Associated Complex that Regulates Sir4 Binding and TPE.

SMC proteins constitute the core members of the Smc5/6, cohesin and condensin complexes. We demonstrate that Smc5/6 is present at telomeres throughout the cell cycle and its association with chromosome ends is dependent on Nse3, a subcomponent of the complex. Cells harboring a temperature sensitive mutant, nse3-1, are defective in Smc5/6 localization to telomeres and have slightly shorter telomeres. Nse3 interacts physically and genetically with two Rap1-binding factors, Rif2 and Sir4. Reduction in telomere-associated Smc5/6 leads to defects in telomere clustering, dispersion of the silencing factor, Sir4, and a loss in transcriptional repression for sub-telomeric genes and non-coding telomeric repeat-containing RNA (TERRA). SIR4 recovery at telomeres is reduced in cells lacking Smc5/6 functionality and vice versa. However, nse3-1/ sir4 Δ double mutants show additive defects for telomere shortening and TPE indicating the contribution of Smc5/6 to telomere homeostasis is only in partial overlap with SIR factor silencing. These findings support a role for Smc5/6 in telomere maintenance that is separate from its canonical role(s) in HR-mediated events during replication and telomere elongation.

Non-Smc element 5 (Nse5) of the Smc5/6 complex interacts with SUMO pathway components.

The Smc5/6 complex in Saccharomyces cerevisiae contains six essential non-Smc elements, Nse1-6. With the exception of Nse2 (also known as Mms21), which is an E3 small ubiquitin-like modifier (SUMO) ligase, very little is understood about the role of these components or their contribution to Smc5/6 functionality. Our characterization of Nse5 establishes a previously unidentified relationship between the Smc5/6 complex and factors of the SUMO pathway. Nse5 physically associates with the E2 conjugating enzyme, Ubc9, where contacts are stabilized by non-covalent interactions with SUMO. SUMO also mediates the interactions between Nse5 and the two PIAS family E3 SUMO ligases, Siz1 and Siz2. Cells carrying the nse5-ts1 allele or lacking either SIZ1 or SIZ2 exhibit a reduction in Smc5 sumoylation upon MMS treatment and demonstrate functional redundancy for SUMO mediated events in the presence of DNA damage. Overall, given the extensive connection between Nse5 and components of the SUMO pathway, we speculate that one function of the Smc5/6 complex might be as a scaffold center to enable sumoylation events in budding yeast.

PolySUMOylation by Siz2 and Mms21 triggers relocation of DNA breaks to nuclear pores through the Slx5/Slx8 STUbL.

High-resolution imaging shows that persistent DNA damage in budding yeast localizes in distinct perinuclear foci for repair. The signals that trigger DNA double-strand break (DSB) relocation or determine their destination are unknown. We show here that DSB relocation to the nuclear envelope depends on SUMOylation mediated by the E3 ligases Siz2 and Mms21. In G1, a polySUMOylation signal deposited coordinately by Mms21 and Siz2 recruits the SUMO targeted ubiquitin ligase Slx5/Slx8 to persistent breaks. Both Slx5 and Slx8 are necessary for damage relocation to nuclear pores. When targeted to an undamaged locus, however, Slx5 alone can mediate relocation in G1-phase cells, bypassing the requirement for polySUMOylation. In contrast, in S-phase cells, monoSUMOylation mediated by the Rtt107-stabilized SMC5/6-Mms21 E3 complex drives DSBs to the SUN domain protein Mps3 in a manner independent of Slx5. Slx5/Slx8 and binding to pores favor repair by ectopic break-induced replication and imprecise end-joining.

The Mre11-Rad50-Xrs2 complex is required for yeast DNA postreplication repair.

Yeast DNA postreplication repair (PRR) bypasses replication-blocking lesions to prevent damage-induced cell death. PRR employs two different mechanisms to bypass damaged DNA, namely translesion synthesis (TLS) and error-free PRR, which are regulated via sequential ubiquitination of proliferating cell nuclear antigen (PCNA). We previously demonstrated that error-free PRR utilizes homologous recombination to facilitate template switching. To our surprise, genes encoding the Mre11-Rad50-Xrs2 (MRX) complex, which are also required for homologous recombination, are epistatic to TLS mutations. Further genetic analyses indicated that two other nucleases involved in double-strand end resection, Sae2 and Exo1, are also variably required for efficient lesion bypass. The involvement of the above genes in TLS and/or error-free PRR could be distinguished by the mutagenesis assay and their differential effects on PCNA ubiquitination. Consistent with the observation that the MRX complex is required for both branches of PRR, the MRX complex was found to physically interact with Rad18 in vivo. In light of the distinct and overlapping activities of the above nucleases in the resection of double-strand breaks, we propose that the interplay between distinct single-strand nucleases dictate the preference between TLS and error-free PRR for lesion bypass.

The C-terminus of Nej1 is critical for nuclear localization and non-homologous end-joining.

Nej1 is an essential factor in the non-homologous end-joining (NHEJ) pathway and interacts with the DNA ligase complex, Lif1-Dnl4, through interactions with Lif1. We have mapped K331-V338 in the C-terminal region of Nej1 to be critical for its functionality during repair. Truncation and alanine scanning mutagenesis have been used to identify a motif in Nej1, KKRK (331-334), which is important for both nuclear targeting and NHEJ repair after localization. We have identified F335-V338 to be important for proper interaction with Lif1, however this region is not required for Nej1 recruitment to HO endonuclease-induced DNA double-strand breaks in vivo. Phenylalanine at position 335 is particularly important for the role of Nej1 in repair and the loss of association between Nej1 and Lif1 correlates with a decrease in cell survival upon either transient or continuous HO expression in nej1 mutants.

Cohesin association to replication sites depends on rad50 and promotes fork restart.

The cohesin complex holds together newly replicated chromatids and is involved in diverse pathways that preserve genome integrity. We show that in budding yeast, cohesin is transiently recruited to active replication origins, and it spreads along DNA as forks progress. When DNA synthesis is impeded, cohesin accumulates at replication sites and is critical for the recovery of stalled forks. Cohesin enrichment at replication forks does not depend on γH2A(X) formation, which differs from its loading requirements at DNA double-strand breaks (DSBs). However, cohesin localization is largely reduced in rad50Δ mutants and in cells lacking both Mec1 and Tel1 checkpoint kinases. Interestingly, cohesin loading at replication sites depends on the structural features of Rad50 that are important for bridging sister chromatids, including the CXXC hook domain and the length of the coiled-coil extensions. Together, these data reveal a function for cohesin in the maintenance of genome integrity during S phase.

During replication stress, non-SMC element 5 (NSE5) is required for Smc5/6 protein complex functionality at stalled forks.

The Smc5/6 complex belongs to the SMC (structural maintenance of chromosomes) family, which also includes cohesin and condensin. In Saccharomyces cerevisiae, the Smc5/6 complex contains six essential non-Smc elements, Nse1-6. Very little is known about how these additional elements contribute to complex function except for Nse2/Mms21, which is an E3 small ubiquitin-like modifier (SUMO) ligase important for Smc5 sumoylation. Characterization of two temperature-sensitive mutants, nse5-ts1 and nse5-ts2, demonstrated the importance of Nse5 within the Smc5/6 complex for its stability and functionality at forks during hydroxyurea-induced replication stress. Both NSE5 alleles showed a marked reduction in Smc5 sumoylation to levels lower than those observed with mms21-11, a mutant of Mms21 that is deficient in SUMO ligase activity. However, a phenotypic comparison of nse5-ts1 and nse5-ts2 revealed a separation of importance between Smc5 sumoylation and the function of the Smc5/6 complex during replication. Only cells carrying the nse5-ts1 allele exhibited defects such as dissociation of the replisome from stalled forks, formation of fork-associated homologous recombination intermediates, and hydroxyurea sensitivity that is additive with mms21-11. These defects are attributed to a failure in Smc5/6 localization to forks in nse5-ts1 cells. Overall, these data support the premise that Nse5 is important for vital interactions between components within the Smc5/6 complex, and for its functionality during replication stress.