Role of Microhomology in the Repair of Double-Stranded DNA breaks in Saccharomyces cerevisiae

Role of Microhomology in the Repair of Double-Stranded DNA breaks in Saccharomyces cerevisiae | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Role of Microhomology in the Repair of Double-Stranded DNA breaks in Saccharomyces cerevisiae Stacey Nguyen, Jie Tang, Kevin Truong, Amanda Wang, Ahmad Karabala, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8982208/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Microhomology-mediated end-joining (MMEJ) is an error-prone DNA double-strand break repair pathway. The high mutation and genome rearrangement rates associated with MMEJ contribute to genetic plasticity but may also induce malignancy, generating significant research interest. Previous MMEJ studies have examined the use of microhomologies (MH) on either side of a double-strand break to facilitate repair. However, little evidence shows the involvement of MH in double-strand break repair (DSBR). Using Saccharomyces cerevisiae , we demonstrate the use of MH in DSBR of an induced double-strand break, which is influenced by MH length and continuity. In contrast, MH did not facilitate break-induced replication under similar circumstances. Although the frequency of homologous recombination using MH is comparatively low, it still represents a potential pathway for genome rearrangements and loss of heterozygosity in regions containing short repetitive sequences. Biological sciences/Cancer Biological sciences/Genetics Biological sciences/Molecular biology Microhomology Microhomology-mediated end-joining DNA repair Saccharomyces cerevisiae homologous recombination Figures Figure 1 Figure 2 Figure 3 Background Everyday our cells are exposed to external and internal assaults which can result in DNA damage throughout our genome [ 1 ]. If DNA damage is left unrepaired, it can lead to mutations or rearrangements that can impact the function of important genes (e.g. tumor suppressor genes), which in turn can have deleterious consequences [ 2 ]. Of particular importance is repairing double-strand breaks (DSBs), which compromise genomic integrity and lead to cell death if not repaired [ 3 ]. Therefore, multiple mechanisms for repairing DSBs have evolved, including double-strand break repair (DSBR), which uses the undamaged sister chromatid or homologous chromosome for repair, classical non-homologous end joining (C-NHEJ) via Ku70/Ku80 protein binding and re-ligation of broken ends with DNA ligase, break induced replication (BIR) that uses homology with an intact chromosome to invade and copy the genetic material to the chromosome end, and microhomology-mediated end-joining (MMEJ) via microhomologies near the break-site [ 3 – 4 ]. Previous work has demonstrated that MMEJ facilitates annealing of 3’ single-stranded DNA overhangs using microhomologies that flank the double-stranded break, resulting in deletion of intervening sequences [ 5 – 7 ]. DSBR is a “high fidelity” repair pathway because the repair itself is performed using an intact template (sister chromatid or homologous chromosome) [ 3 – 4 ]. Conversely, MMEJ involves locating and aligning microhomologies, that may be mismatched, across from a DSB without a template. Therefore, MMEJ results in deletion of the intervening sequence between the microhomologies and possible base substitutions if the aligned microhomologies is imperfect [ 5 – 7 ]. Despite the chromosomal rearrangements associated with MMEJ, it serves as a valuable alternative repair pathway when conditions for DSBR and C-NHEJ are not satisfied [ 8 ]. Defining the genetic control and influence of microhomology lengths has been a major focus of MMEJ studies [ 5 – 11 ]. However, the mechanism for repair in different organisms has largely been focused on an end-joining model similar to single-strand annealing. Understanding how microhomologies are used in other DNA repair contexts has implications for tumor development and treatment. Materials and Methods Yeast Strains Diploid S. cerevisiae isogenic to W303-1A and wildtype for RAD5 , were genetically engineered to possess varying lengths of shared microhomology (MH) between two truncated his3 alleles ( his3Δ3’ and his3Δ5’ ) at the HIS3 locus on chromosome XV of homologous chromosomes (Supplemental Table 1) for DSBR or at the HIS3 locus on chromosome XV ( his3Δ3’ ) and LEU2 locus on chromosome III ( his3Δ5’ ) for BIR. The MH was either complete MH (16 bp, 20 bp, or 25 bp) or with a 2-base pair mismatch within the MH (14. 2 .2 bp, 14. 2 .4 bp, or 14. 2 .9 bp) and adjacent to a 117-basepair HO endonuclease recognition sequence [ 6 ]. A his3Δ3’ allele sharing 311 bp of homology with his3Δ5’ was used as a comparison and expression of HO endonuclease was controlled by a galactose promoter located at the trp1 locus ( trp1::GAL1-HO-KANMX ) on chromosome IV [ 9 ]. All diploids possessed the MATa-inc and MAT𝝰-inc alleles that are not cut by HO endonuclease. Double-Strand Break Repair and Break Induced Replication Assay Single yeast colonies of the appropriate genotype were grown overnight at 30 ℃ in two milliliters of media containing 1% yeast extract, 2% peptone, and 2% raffinose. Following overnight growth, galactose was added to the culture to a final concentration of 2% and incubated an additional four hours at 30 ℃. The addition of galactose induced the expression of HO endonuclease that targeted the HO recognition sequence next to the his3Δ3’ allele creating a DSB. Following HO expression and DSB formation, repair using specific MH or 311 bp next to the break was monitored following serial dilution and plating onto nutrient rich media, YPD, and selective media, SD-His, plates to determine the frequency of HIS+ recombinants. The median frequency of His+ recombinants +/- 95% confidence interval was determined for each MH length and 311 bp. Results To address utilization of microhomologies in DSBR, we evaluated the frequency of repair when microhomologies of varying lengths and continuity were introduced into the 3’-end of a truncated his3Δ3’ allele on chromosome XV, which also contains an HO endonuclease recognition sequence (Fig. 1A). The addition of galactose induces expression of HO endonuclease to generate a DSB next to the his3Δ3’ allele. Repair of the DSB proceeds through invasion of the broken molecule with the intact homologous chromosome containing a his3Δ5’ allele and subsequent repair. Previous work showed the repair product (His+ recombinants or his3Δ5’ recombinants) is determined by which end of the broken DNA molecule is utilized for invasion, left-end or right-end, to produce colonies with distinct colors, red or pale, respectively (Fig. 1A and 1B) [ 12 ]. The generation of His+ recombinants used microhomologies of varying lengths and contained no mismatches (16 bp, 20 bp, or 25 bp) or a 2-bp mismatch (14-2-2 bp, 14-2-4 bp, or 14-2-9 bp), which was compared to a homology length of 311 bp to determine the relative repair frequencies (Fig. 1C). Repair utilizing microhomology required left-end invasion following DSB formation, producing a His+ recombinant and red colony. The repair efficiency of complete microhomology sequence lengths, 16, 20, and 25 bp were similar to each other, showing that small changes in microhomology length are insufficient to influence overall repair efficiency (Fig. 2A, Table 1 ). The addition of a 2-bp mismatch within the microhomology sequences resulted in a significant decrease of 24-fold (14-2-9 vs 25 bp), 43-fold (14-2-4 vs 20 bp), or 433-fold (14-2-2 vs 16 bp) in the efficiency of repair compared to the complete homology (Fig. 2A, Table 1 ). This demonstrates the compounding negative impact of mismatches, even as microhomologies length increases. However, DSB repair using microhomology is at least 1250-fold lower compared to the 311 bp sequence regardless of homology length or continuity (Fig. 2A, Table 1 ). In contrast to microhomology directed left-end invasion and repair, right-end invasion uses greater than 1000 bp of homology to generate a his3Δ5’ recombinant that appears as a pale colony on YPD plates (Fig. 1B). As expected, the frequency of pale colonies observed is significantly higher than His+ recombinants for all genotypes tested (Fig. 2B). Examination of the pale colony frequency in cells possessing varying microhomologies showed a significant increase when compared to 311 bp (Fig. 2B, Table 1 ). A consistent increase of 13–25% in pale colony frequency was observed for all cells possessing microhomologies compared to the longer 311 bp of homology. This suggests that the sequence context on either side of a DSB influences which end would be utilized for invasion of an intact homologous sequence. Taken together, the above results demonstrate that microhomologies next to a DSB can both facilitate and influence overall DSBR using an intact homologous chromosome and the efficiency of repair is dependent on the length and continuity of microhomology, similar to previous studies [ 5 – 11 ]. Break-induced replication (BIR) is a pathway which utilizes homologous sequences on one end of a DSB to invade an intact chromosome and copy the entire chromosome arm through the telomere [ 3 ]. In our DSBR system, BIR would also result in His+ recombinants following DSB formation and left-ended invasion. To test this, we used the same his3Δ3’ substrates with 20, 25, and 311 bp of homology, but moved the his3Δ5’ to the LEU2 locus on chromosome III to specifically examine ectopic BIR (Fig. 3A). Our results show ectopic BIR can occur when 311 bp of shared homology exists between the truncated his3 sequences (Fig. 3B). However, we were unable to detect a single His+ recombinant when either 20 bp or 25 bp of microhomology was used in ectopic BIR (Fig. 3B). This provides evidence that repair using microhomologies is more likely to occur in specific repair pathways. Discussion While MMEJ is not the preferred repair pathway for DSBs, it is likely utilized under certain conditions that include specific homology length of 5–30 bp, the phase of the cell cycle, protein expression levels, and the continuity of homology [ 2 – 3 , 5 – 7 ]. Moreover, there is likely an ongoing competition between various repair pathways, including MMEJ, DSBR, and NHEJ. Using a competition repair substrate for MMEJ and DSBR, Lan et al. showed that in mammalian cells, MMEJ with short end resection occurs at 10–20% of the DSBR rate, when both DSBR and NHEJ are available [ 5 ]. Despite the presence of microhomology and conditions favoring MMEJ, other repair pathways like NHEJ and DSBR could still prevail if they are more efficient or if the proteins involved in these pathways are present in higher quantities. This is especially applicable when the DNA breaks are suitable for multiple repair mechanisms. In this study, we established the use of microhomologies, typical of the MMEJ pathway in S. cerevisiae , in the repair of DSBs using an intact homologous chromosome. Our findings show no increase in MMEJ frequency for 16 to 25 bp, which contrasts the findings of Villareal et al., Lee et al., and Meyer et al., who observed a correlation between increasing lengths of microhomology and MMEJ efficiency [ 9 – 11 ]. Notably, however, when sequences containing di-nucleotide mismatches (14-2-2, 14-2-4, and 14-2-9 bp) were inserted within microhomologies, there was a rise in MMEJ repair frequency with increasing length, but still lower compared to the complete microhomology counterpart (16 vs. 14-2-2, 20 vs. 14-2-4, and 25 vs. 14-2-9 bp). There are several possibilities that could explain our observations on how microhomology length and continuity influence repair frequency. First, MMEJ relies on short microhomologies to align the broken DNA ends during repair. In the presence of mismatches, longer microhomologies may allow for greater stability of the repair intermediate. Mismatches, especially at shorter microhomology lengths, may create structural instability that impedes efficient engagement of DNA polymerases [ 9 , 13 ]. By increasing the length of the microhomology, the system may "compensate" for the mismatch by stabilizing the repair ends, which become more favorable for extension by DNA polymerase. In addition, MMEJ occurs in the presence of mismatches suggesting a tolerance of mismatches, particularly when microhomology is longer [ 9 – 11 ]. Therefore, an increase in microhomology length may reduce the negative effects of mismatches, making MMEJ more efficient as the mismatch burden is distributed across a larger homology region, allowing more successful repairs. Both MMEJ and DSBR share the initial end resection step in DSB repair and rely on some common repair proteins. However, DSBR also requires additional factors, such as Rad51 and Rad52, which may be more available or preferentially recruited during specific cell cycle stages [ 9 , 14 – 19 ]. Another protein, RPA plays a vital role in HR, binding to single-stranded DNA of 30 nucleotides or longer to prevent degradation by nucleases and facilitate loading of the Rad51 recombinase by Rad52 [ 15 ]. Previous work showed that Rad52, Rad51, and RPA may antagonize MMEJ by promoting DSBR or preventing the spontaneous annealing of microhomology sequences bound by RPA [ 9 , 16 – 17 ]. This could make DSBR more likely to be favored over MMEJ, which may explain our findings that His+ recombinants occur at a higher frequency when 311 bp of homology, typical of DSBR, is used for repair. Ultimately, repair pathway competition is complex and is influenced by homology length and the presence of specific proteins. Advancements in the study of DSB repair mechanisms pertaining to DSBR and MMEJ will serve as a cornerstone for progress in cancer treatment research and development. Research focused specifically on MMEJ is particularly crucial for developing treatments targeting DSBR-deficient cancer lines. The major types of DSBR-deficient cancers include BRCA1/BRCA2-mutated triple-negative breast cancers (TNBC), high-grade serous ovarian carcinoma (HGSOC), BRCA-mutated metastatic castration-resistant prostate cancer (mCRPC), BRCA-mutated pancreatic adenocarcinoma, certain subtypes of endometrial cancer, and some cases of small cell lung cancer (SCLC) to name a few [ 20 ]. Our results suggest that, in favorable conditions taking into account favorable microhomology lengths and continuity, DSBR-deficient cell lines have the ability to rely on MMEJ as an alternate pathway for repair, highlighting the critical need to develop treatments that specifically target and disrupt this mechanism – impeding tumor survivability. In fact, Creeden et al. highlight that examples of this approach have already been extensively studied and applied, such as through pharmacological drugs acting as Poly (ADP-ribose) polymerase inhibitors (PARPi) [ 21 ]. Most notably used in BRCA1/BRCA2 dependent breast cancer identified to be DSBR-deficient, PARPi suppresses the pathway for single-strand break repair. This strategy of synthetic lethality limits the available repair pathways for a cancer cell, effectively forcing the cell to undergo apoptosis, thereby achieving clinical goals in cancer treatment [ 22 – 23 ]. Additionally, previous studies have demonstrated that MMEJ may be utilized even when DSBR and other repair pathways are available [ 5 – 11 ]. Thus, even for cancers not considered to be DSBR-deficient, MMEJ remains as a critical repair pathway, further underscoring the importance of continued research in this field and its potential for broad relevance across all cancer types. DNA polymerase theta (Polθ), an enzyme found to be responsible for mediating the activation of MMEJ in human cells by assisting the processing of DSBs as one of several proteins involved in early recruitment after identification of a DSB [ 24 ]. This finding has been upheld in studies involving both human DNA cells and S. cerevisiae yeast lines, with the latter studying Pol4, a Polθ equivalent in yeast cells [ 24 – 26 ]. Consequently, forms of cancer that are attributed with Polθ overexpression such as several aggressive types of thyroid cancer have been correlated with poor prognosis [ 25 ]. Yet it remains largely undetermined when, why, and under which conditions Polθ is recruited to initiate MMEJ. Current hypotheses allude that elevated Polθ expression in these tumors may result, at least in part, from an underlying DSBR deficiency. The recognition that DSBR deficiency may be the reason for Polθ upregulation as a survival strategy guides future directions for research of potential pharmaceutical therapies that could target Polθ, thus inhibiting aberrant MMEJ activation altogether. Declarations Ethics approval and consent to participate: Not applicable Consent for publication: Not applicable A vailability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests: Not applicable Funding: This work was supported by California Northstate University, College of Health Sciences. Authors' contributions: SN, JT, KT, and AW conducted all DSBR experiments, collected and analyzed data, and wrote the main manuscript. AK, YC, and SA conducted all BIR experiments and helped write part of manuscript. DM conceived the experiments, supervised the project, edited the manuscript, and created figures and table. All authors reviewed the manuscript and provided critical feedback. Acknowledgements: We would like to acknowledge California Northstate University for providing the facilities and resources to complete this work. References Barnes, D. E. & Lindahl, T. Repair and genetic consequences of endogenous DNA base damage in mammalian cells. Annu. Rev. Genet. 38 , 445–476. https://doi.org/10.1146/annurev.genet.38.072902.092448 (2004). Hakem, R. DNA-damage repair; the good, the bad, and the ugly. EMBO J. 27 (4), 589–605. https://doi.org/10.1038/emboj.2008.15 (2008). Ceccaldi, R., Rondinelli, B. & D'Andrea, A. D. Repair Pathway Choices and Consequences at the Double-Strand Break. Trends Cell Biol. 26 (1), 52–64. https://doi.org/10.1016/j.tcb.2015.07.009 (2016). Symington, L. S., Rothstein, R. & Lisby, M. Mechanisms and regulation of mitotic recombination in Saccharomyces cerevisiae. Genetics 198 (3), 795–835. https://doi.org/10.1534/genetics.114.166140 (2014). Truong, L. N. et al. 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Supplementary Files SupplementalTable1.docx Supplemental Table 1: Saccharomyces cerevisiae strains a All strains are wild type for RAD5 unless indicated. Table11.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8982208","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":601530940,"identity":"44970ed1-db5c-48ab-a636-0c41cda58adb","order_by":0,"name":"Stacey Nguyen","email":"","orcid":"","institution":"California Northstate University","correspondingAuthor":false,"prefix":"","firstName":"Stacey","middleName":"","lastName":"Nguyen","suffix":""},{"id":601530941,"identity":"7b701755-9002-4651-87d3-4ee59c9dd79c","order_by":1,"name":"Jie Tang","email":"","orcid":"","institution":"California Northstate University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Tang","suffix":""},{"id":601530943,"identity":"91cc5e52-8064-4bad-8f6b-9dce30f0655d","order_by":2,"name":"Kevin Truong","email":"","orcid":"","institution":"California Northstate University","correspondingAuthor":false,"prefix":"","firstName":"Kevin","middleName":"","lastName":"Truong","suffix":""},{"id":601530944,"identity":"b2e3c5da-4794-494b-b914-ba4b7e15b172","order_by":3,"name":"Amanda Wang","email":"","orcid":"","institution":"California Northstate University","correspondingAuthor":false,"prefix":"","firstName":"Amanda","middleName":"","lastName":"Wang","suffix":""},{"id":601530945,"identity":"badbbe01-58b9-47f2-ba34-2a877dc4b953","order_by":4,"name":"Ahmad Karabala","email":"","orcid":"","institution":"Kansas Health Science University","correspondingAuthor":false,"prefix":"","firstName":"Ahmad","middleName":"","lastName":"Karabala","suffix":""},{"id":601530947,"identity":"b275fef1-ac38-4dcb-a801-481e93239d37","order_by":5,"name":"Yu-Tung Chen","email":"","orcid":"","institution":"California Northstate University","correspondingAuthor":false,"prefix":"","firstName":"Yu-Tung","middleName":"","lastName":"Chen","suffix":""},{"id":601530948,"identity":"3186ef45-b5c1-4ff3-9d7d-491a2f6163be","order_by":6,"name":"Sohum Acharya","email":"","orcid":"","institution":"California Health Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Sohum","middleName":"","lastName":"Acharya","suffix":""},{"id":601530949,"identity":"ed71c5ae-276a-4b4f-8063-bc1b38269b8d","order_by":7,"name":"Aseel Alkaabi","email":"","orcid":"","institution":"California Northstate University","correspondingAuthor":false,"prefix":"","firstName":"Aseel","middleName":"","lastName":"Alkaabi","suffix":""},{"id":601530951,"identity":"e826722b-cb02-4d34-9add-db880050e598","order_by":8,"name":"Damon Meyer","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYDCCAwgm4wMow4BoLcwwpcRrYZMgSgvf8dOJjwsYtskZHO99VvFzR11iA3vzNgl8WiTP5G42nsFw29jgzHGzm71nDic28Bwrw6vF4EDuNmkehtuJG26ksd1mbDuQ2CCRY4Zfy/m3YC31G+4/YytmbAM6TP4NAS03ILYkGNxgY2NmbGMG2sKDX4vkjbebjXkMbhvOPJPGLNnbdti4jSet2AKfFr7zuRsf81Tcluc7fozxw8+2Otl+9sMbb+DTAnUeA4PCASibjbByKJBvIFrpKBgFo2AUjDQAAAolS8oRJOzlAAAAAElFTkSuQmCC","orcid":"","institution":"California Northstate University","correspondingAuthor":true,"prefix":"","firstName":"Damon","middleName":"","lastName":"Meyer","suffix":""}],"badges":[],"createdAt":"2026-02-27 01:08:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8982208/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8982208/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104069672,"identity":"08768599-821b-42ec-a831-b7d63d462edf","added_by":"auto","created_at":"2026-03-06 11:38:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":545294,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInterchromosomal MMEJ\u003c/strong\u003e \u003cstrong\u003eAssay\u003c/strong\u003e. (A) Recombination between \u003cem\u003ehis3D3’\u003c/em\u003e and \u003cem\u003ehis3D5’\u003c/em\u003e alleles following double-strand break formation results in His+ recombinant red colonies or \u003cem\u003ehis3D5’\u003c/em\u003e pale colonies on YPD. (B) YPD plate showing two colony types (red and pale) following double-strand break repair.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8982208/v1/256fee31386b043ed56bde36.png"},{"id":104069667,"identity":"7eed780a-4b49-49ff-8c84-46f8806def73","added_by":"auto","created_at":"2026-03-06 11:38:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":457176,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHis+ and Pale Recombinant Frequency Using Varying Lengths of Microhomology. \u003c/strong\u003eThe median His+ recombinant (A) or Pale recombinant (B) frequency ± 95% confidence interval was determined from a minimum of ten (10) independent cultures. Varying amounts of microhomology lengths without a mismatch (16 bp, 20 bp, and 25 bp) or with a 2-bp mismatch (14-2-2 bp, 14-2-4 bp, and 14-2-9 bp) shared between \u003cem\u003ehis3D3’\u003c/em\u003e and \u003cem\u003ehis3D5’\u003c/em\u003e alleles were present in all diploid cells. For comparison, homology length typical of homologous recombination (311 bp) was included.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8982208/v1/ccb483a47fd84cf267d1fbc8.png"},{"id":104069973,"identity":"72215617-aee4-45cb-a3ed-1d1e2a43f1f9","added_by":"auto","created_at":"2026-03-06 11:40:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":98105,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicrohomology Does Not Facilitate BIR. \u003c/strong\u003e(A)\u003cstrong\u003e \u003c/strong\u003eEctopic BIR between\u003cem\u003e his3D3’\u003c/em\u003e on chromosome XV and \u003cem\u003ehis3D5’\u003c/em\u003e on chromosome III creates His+ recombinants. (B) The median His+ recombination frequency ± 95% confidence interval for 311 bp and complete microhomology (20 bp and 25 bp) shared between \u003cem\u003ehis3D3’\u003c/em\u003e and \u003cem\u003ehis3D5’\u003c/em\u003e alleles. A minimum of nine (9) independent diploid cultures were used for each homology length.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8982208/v1/eba97e5add778fd04b463ecf.png"},{"id":105564826,"identity":"66470b16-85f5-445a-aea2-fad60a934edc","added_by":"auto","created_at":"2026-03-27 12:50:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1466084,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8982208/v1/06122b5a-ac15-48f2-936c-1b302387a717.pdf"},{"id":104070090,"identity":"74d7e125-b255-4488-b2ce-c20dcc1a4af0","added_by":"auto","created_at":"2026-03-06 11:40:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":27897,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Table 1: \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSaccharomyces cerevisiae \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003estrains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eAll strains are wild type for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eRAD5 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eunless indicated.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"SupplementalTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8982208/v1/49dfc3ec4fcb5618021f52c3.docx"},{"id":104069680,"identity":"3d9702f9-992a-4b5a-978f-0efee7f7843b","added_by":"auto","created_at":"2026-03-06 11:38:15","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":19067,"visible":true,"origin":"","legend":"","description":"","filename":"Table11.docx","url":"https://assets-eu.researchsquare.com/files/rs-8982208/v1/2de365c240fa3e7b261cd16a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Role of Microhomology in the Repair of Double-Stranded DNA breaks in Saccharomyces cerevisiae","fulltext":[{"header":"Background","content":"\u003cp\u003eEveryday our cells are exposed to external and internal assaults which can result in DNA damage throughout our genome [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. If DNA damage is left unrepaired, it can lead to mutations or rearrangements that can impact the function of important genes (e.g. tumor suppressor genes), which in turn can have deleterious consequences [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Of particular importance is repairing double-strand breaks (DSBs), which compromise genomic integrity and lead to cell death if not repaired [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, multiple mechanisms for repairing DSBs have evolved, including double-strand break repair (DSBR), which uses the undamaged sister chromatid or homologous chromosome for repair, classical non-homologous end joining (C-NHEJ) via Ku70/Ku80 protein binding and re-ligation of broken ends with DNA ligase, break induced replication (BIR) that uses homology with an intact chromosome to invade and copy the genetic material to the chromosome end, and microhomology-mediated end-joining (MMEJ) via microhomologies near the break-site [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Previous work has demonstrated that MMEJ facilitates annealing of 3\u0026rsquo; single-stranded DNA overhangs using microhomologies that flank the double-stranded break, resulting in deletion of intervening sequences [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDSBR is a \u0026ldquo;high fidelity\u0026rdquo; repair pathway because the repair itself is performed using an intact template (sister chromatid or homologous chromosome) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Conversely, MMEJ involves locating and aligning microhomologies, that may be mismatched, across from a DSB without a template. Therefore, MMEJ results in deletion of the intervening sequence between the microhomologies and possible base substitutions if the aligned microhomologies is imperfect [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Despite the chromosomal rearrangements associated with MMEJ, it serves as a valuable alternative repair pathway when conditions for DSBR and C-NHEJ are not satisfied [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDefining the genetic control and influence of microhomology lengths has been a major focus of MMEJ studies [\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9 CR10\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, the mechanism for repair in different organisms has largely been focused on an end-joining model similar to single-strand annealing. Understanding how microhomologies are used in other DNA repair contexts has implications for tumor development and treatment.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eYeast Strains\u003c/h2\u003e \u003cp\u003eDiploid \u003cem\u003eS. cerevisiae\u003c/em\u003e isogenic to W303-1A and wildtype for \u003cem\u003eRAD5\u003c/em\u003e, were genetically engineered to possess varying lengths of shared microhomology (MH) between two truncated \u003cem\u003ehis3\u003c/em\u003e alleles (\u003cem\u003ehis3Δ3\u0026rsquo;\u003c/em\u003e and \u003cem\u003ehis3Δ5\u0026rsquo;\u003c/em\u003e) at the \u003cem\u003eHIS3\u003c/em\u003e locus on chromosome XV of homologous chromosomes (Supplemental Table\u0026nbsp;1) for DSBR or at the \u003cem\u003eHIS3\u003c/em\u003e locus on chromosome XV (\u003cem\u003ehis3Δ3\u0026rsquo;\u003c/em\u003e) and LEU2 locus on chromosome III (\u003cem\u003ehis3Δ5\u0026rsquo;\u003c/em\u003e) for BIR. The MH was either complete MH (16 bp, 20 bp, or 25 bp) or with a 2-base pair mismatch within the MH (14.\u003cb\u003e2\u003c/b\u003e.2 bp, 14.\u003cb\u003e2\u003c/b\u003e.4 bp, or 14.\u003cb\u003e2\u003c/b\u003e.9 bp) and adjacent to a 117-basepair HO endonuclease recognition sequence [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. A \u003cem\u003ehis3Δ3\u0026rsquo;\u003c/em\u003e allele sharing 311 bp of homology with \u003cem\u003ehis3Δ5\u0026rsquo;\u003c/em\u003e was used as a comparison and expression of HO endonuclease was controlled by a galactose promoter located at the \u003cem\u003etrp1\u003c/em\u003e locus (\u003cem\u003etrp1::GAL1-HO-KANMX\u003c/em\u003e) on chromosome IV [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. All diploids possessed the \u003cem\u003eMATa-inc\u003c/em\u003e and \u003cem\u003eMAT\u0026#120688;-inc\u003c/em\u003e alleles that are not cut by HO endonuclease.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDouble-Strand Break Repair and Break Induced Replication Assay\u003c/h3\u003e\n\u003cp\u003eSingle yeast colonies of the appropriate genotype were grown overnight at 30 ℃ in two milliliters of media containing 1% yeast extract, 2% peptone, and 2% raffinose. Following overnight growth, galactose was added to the culture to a final concentration of 2% and incubated an additional four hours at 30 ℃. The addition of galactose induced the expression of HO endonuclease that targeted the HO recognition sequence next to the \u003cem\u003ehis3Δ3\u0026rsquo;\u003c/em\u003e allele creating a DSB. Following HO expression and DSB formation, repair using specific MH or 311 bp next to the break was monitored following serial dilution and plating onto nutrient rich media, YPD, and selective media, SD-His, plates to determine the frequency of HIS+ recombinants. The median frequency of His+ recombinants +/- 95% confidence interval was determined for each MH length and 311 bp.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTo address utilization of microhomologies in DSBR, we evaluated the frequency of repair when microhomologies of varying lengths and continuity were introduced into the 3\u0026rsquo;-end of a truncated \u003cem\u003ehis3\u0026Delta;3\u0026rsquo;\u003c/em\u003e allele on chromosome XV, which also contains an HO endonuclease recognition sequence (Fig. 1A). The addition of galactose induces expression of HO endonuclease to generate a DSB next to the \u003cem\u003ehis3\u0026Delta;3\u0026rsquo;\u003c/em\u003e allele. Repair of the DSB proceeds through invasion of the broken molecule with the intact homologous chromosome containing a \u003cem\u003ehis3\u0026Delta;5\u0026rsquo;\u003c/em\u003e allele and subsequent repair. Previous work showed the repair product (His+ recombinants or \u003cem\u003ehis3\u0026Delta;5\u0026rsquo;\u003c/em\u003e recombinants) is determined by which end of the broken DNA molecule is utilized for invasion, left-end or right-end, to produce colonies with distinct colors, red or pale, respectively (Fig. 1A and 1B) [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe generation of His+ recombinants used microhomologies of varying lengths and contained no mismatches (16 bp, 20 bp, or 25 bp) or a 2-bp mismatch (14-2-2 bp, 14-2-4 bp, or 14-2-9 bp), which was compared to a homology length of 311 bp to determine the relative repair frequencies (Fig. 1C). Repair utilizing microhomology required left-end invasion following DSB formation, producing a His+ recombinant and red colony. The repair efficiency of complete microhomology sequence lengths, 16, 20, and 25 bp were similar to each other, showing that small changes in microhomology length are insufficient to influence overall repair efficiency (Fig. 2A, Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The addition of a 2-bp mismatch within the microhomology sequences resulted in a significant decrease of 24-fold (14-2-9 vs 25 bp), 43-fold (14-2-4 vs 20 bp), or 433-fold (14-2-2 vs 16 bp) in the efficiency of repair compared to the complete homology (Fig. 2A, Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). This demonstrates the compounding negative impact of mismatches, even as microhomologies length increases. However, DSB repair using microhomology is at least 1250-fold lower compared to the 311 bp sequence regardless of homology length or continuity (Fig. 2A, Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eIn contrast to microhomology directed left-end invasion and repair, right-end invasion uses greater than 1000 bp of homology to generate a \u003cem\u003ehis3\u0026Delta;5\u0026rsquo;\u003c/em\u003e recombinant that appears as a pale colony on YPD plates (Fig. 1B). As expected, the frequency of pale colonies observed is significantly higher than His+ recombinants for all genotypes tested (Fig. 2B). Examination of the pale colony frequency in cells possessing varying microhomologies showed a significant increase when compared to 311 bp (Fig. 2B, Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). A consistent increase of 13\u0026ndash;25% in pale colony frequency was observed for all cells possessing microhomologies compared to the longer 311 bp of homology. This suggests that the sequence context on either side of a DSB influences which end would be utilized for invasion of an intact homologous sequence. Taken together, the above results demonstrate that microhomologies next to a DSB can both facilitate and influence overall DSBR using an intact homologous chromosome and the efficiency of repair is dependent on the length and continuity of microhomology, similar to previous studies [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eBreak-induced replication (BIR) is a pathway which utilizes homologous sequences on one end of a DSB to invade an intact chromosome and copy the entire chromosome arm through the telomere [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]. In our DSBR system, BIR would also result in His+ recombinants following DSB formation and left-ended invasion. To test this, we used the same \u003cem\u003ehis3\u0026Delta;3\u0026rsquo;\u003c/em\u003e substrates with 20, 25, and 311 bp of homology, but moved the \u003cem\u003ehis3\u0026Delta;5\u0026rsquo;\u003c/em\u003e to the \u003cem\u003eLEU2\u003c/em\u003e locus on chromosome III to specifically examine ectopic BIR (Fig. 3A). Our results show ectopic BIR can occur when 311 bp of shared homology exists between the truncated \u003cem\u003ehis3\u003c/em\u003e sequences (Fig. 3B). However, we were unable to detect a single His+ recombinant when either 20 bp or 25 bp of microhomology was used in ectopic BIR (Fig. 3B). This provides evidence that repair using microhomologies is more likely to occur in specific repair pathways.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWhile MMEJ is not the preferred repair pathway for DSBs, it is likely utilized under certain conditions that include specific homology length of 5\u0026ndash;30 bp, the phase of the cell cycle, protein expression levels, and the continuity of homology [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Moreover, there is likely an ongoing competition between various repair pathways, including MMEJ, DSBR, and NHEJ. Using a competition repair substrate for MMEJ and DSBR, Lan et al. showed that in mammalian cells, MMEJ with short end resection occurs at 10\u0026ndash;20% of the DSBR rate, when both DSBR and NHEJ are available [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Despite the presence of microhomology and conditions favoring MMEJ, other repair pathways like NHEJ and DSBR could still prevail if they are more efficient or if the proteins involved in these pathways are present in higher quantities. This is especially applicable when the DNA breaks are suitable for multiple repair mechanisms.\u003c/p\u003e \u003cp\u003eIn this study, we established the use of microhomologies, typical of the MMEJ pathway in \u003cem\u003eS. cerevisiae\u003c/em\u003e, in the repair of DSBs using an intact homologous chromosome. Our findings show no increase in MMEJ frequency for 16 to 25 bp, which contrasts the findings of Villareal et al., Lee et al., and Meyer et al., who observed a correlation between increasing lengths of microhomology and MMEJ efficiency [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Notably, however, when sequences containing di-nucleotide mismatches (14-2-2, 14-2-4, and 14-2-9 bp) were inserted within microhomologies, there was a rise in MMEJ repair frequency with increasing length, but still lower compared to the complete microhomology counterpart (16 vs. 14-2-2, 20 vs. 14-2-4, and 25 vs. 14-2-9 bp).\u003c/p\u003e \u003cp\u003eThere are several possibilities that could explain our observations on how microhomology length and continuity influence repair frequency. First, MMEJ relies on short microhomologies to align the broken DNA ends during repair. In the presence of mismatches, longer microhomologies may allow for greater stability of the repair intermediate. Mismatches, especially at shorter microhomology lengths, may create structural instability that impedes efficient engagement of DNA polymerases [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. By increasing the length of the microhomology, the system may \"compensate\" for the mismatch by stabilizing the repair ends, which become more favorable for extension by DNA polymerase. In addition, MMEJ occurs in the presence of mismatches suggesting a tolerance of mismatches, particularly when microhomology is longer [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Therefore, an increase in microhomology length may reduce the negative effects of mismatches, making MMEJ more efficient as the mismatch burden is distributed across a larger homology region, allowing more successful repairs.\u003c/p\u003e \u003cp\u003eBoth MMEJ and DSBR share the initial end resection step in DSB repair and rely on some common repair proteins. However, DSBR also requires additional factors, such as Rad51 and Rad52, which may be more available or preferentially recruited during specific cell cycle stages [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Another protein, RPA plays a vital role in HR, binding to single-stranded DNA of 30 nucleotides or longer to prevent degradation by nucleases and facilitate loading of the Rad51 recombinase by Rad52 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Previous work showed that Rad52, Rad51, and RPA may antagonize MMEJ by promoting DSBR or preventing the spontaneous annealing of microhomology sequences bound by RPA [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This could make DSBR more likely to be favored over MMEJ, which may explain our findings that His+ recombinants occur at a higher frequency when 311 bp of homology, typical of DSBR, is used for repair. Ultimately, repair pathway competition is complex and is influenced by homology length and the presence of specific proteins.\u003c/p\u003e \u003cp\u003eAdvancements in the study of DSB repair mechanisms pertaining to DSBR and MMEJ will serve as a cornerstone for progress in cancer treatment research and development. Research focused specifically on MMEJ is particularly crucial for developing treatments targeting DSBR-deficient cancer lines. The major types of DSBR-deficient cancers include BRCA1/BRCA2-mutated triple-negative breast cancers (TNBC), high-grade serous ovarian carcinoma (HGSOC), BRCA-mutated metastatic castration-resistant prostate cancer (mCRPC), BRCA-mutated pancreatic adenocarcinoma, certain subtypes of endometrial cancer, and some cases of small cell lung cancer (SCLC) to name a few [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Our results suggest that, in favorable conditions taking into account favorable microhomology lengths and continuity, DSBR-deficient cell lines have the ability to rely on MMEJ as an alternate pathway for repair, highlighting the critical need to develop treatments that specifically target and disrupt this mechanism \u0026ndash; impeding tumor survivability. In fact, Creeden et al. highlight that examples of this approach have already been extensively studied and applied, such as through pharmacological drugs acting as Poly (ADP-ribose) polymerase inhibitors (PARPi) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Most notably used in BRCA1/BRCA2 dependent breast cancer identified to be DSBR-deficient, PARPi suppresses the pathway for single-strand break repair. This strategy of synthetic lethality limits the available repair pathways for a cancer cell, effectively forcing the cell to undergo apoptosis, thereby achieving clinical goals in cancer treatment [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Additionally, previous studies have demonstrated that MMEJ may be utilized even when DSBR and other repair pathways are available [\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9 CR10\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Thus, even for cancers not considered to be DSBR-deficient, MMEJ remains as a critical repair pathway, further underscoring the importance of continued research in this field and its potential for broad relevance across all cancer types.\u003c/p\u003e \u003cp\u003eDNA polymerase theta (Polθ), an enzyme found to be responsible for mediating the activation of MMEJ in human cells by assisting the processing of DSBs as one of several proteins involved in early recruitment after identification of a DSB [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This finding has been upheld in studies involving both human DNA cells and \u003cem\u003eS. cerevisiae\u003c/em\u003e yeast lines, with the latter studying Pol4, a Polθ equivalent in yeast cells [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Consequently, forms of cancer that are attributed with Polθ overexpression such as several aggressive types of thyroid cancer have been correlated with poor prognosis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Yet it remains largely undetermined when, why, and under which conditions Polθ is recruited to initiate MMEJ. Current hypotheses allude that elevated Polθ expression in these tumors may result, at least in part, from an underlying DSBR deficiency. The recognition that DSBR deficiency may be the reason for Polθ upregulation as a survival strategy guides future directions for research of potential pharmaceutical therapies that could target Polθ, thus inhibiting aberrant MMEJ activation altogether.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate: Not applicable\u003c/p\u003e\n\u003cp\u003eConsent for publication:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003evailability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003eCompeting interests: Not applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunding: This work was supported by California Northstate University, College of Health Sciences.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions: SN, JT, KT, and AW conducted all DSBR experiments, collected and analyzed data, and wrote the main manuscript. AK, YC, and SA conducted all BIR experiments and helped write part of manuscript. DM conceived the experiments, supervised the project, edited the manuscript, and created figures and table. All authors reviewed the manuscript and provided critical feedback.\u003c/p\u003e\n\u003cp\u003eAcknowledgements: We would like to acknowledge California Northstate University for providing the facilities and resources to complete this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBarnes, D. E. \u0026amp; Lindahl, T. Repair and genetic consequences of endogenous DNA base damage in mammalian cells. \u003cem\u003eAnnu. Rev. 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The high mutation and genome rearrangement rates associated with MMEJ contribute to genetic plasticity but may also induce malignancy, generating significant research interest. Previous MMEJ studies have examined the use of microhomologies (MH) on either side of a double-strand break to facilitate repair. However, little evidence shows the involvement of MH in double-strand break repair (DSBR). Using \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, we demonstrate the use of MH in DSBR of an induced double-strand break, which is influenced by MH length and continuity. In contrast, MH did not facilitate break-induced replication under similar circumstances. Although the frequency of homologous recombination using MH is comparatively low, it still represents a potential pathway for genome rearrangements and loss of heterozygosity in regions containing short repetitive sequences.\u003c/p\u003e","manuscriptTitle":"Role of Microhomology in the Repair of Double-Stranded DNA breaks in Saccharomyces cerevisiae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-06 11:38:01","doi":"10.21203/rs.3.rs-8982208/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"36592c9c-40f6-4940-8189-eb6fe1bf80b1","owner":[],"postedDate":"March 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":64015085,"name":"Biological sciences/Cancer"},{"id":64015086,"name":"Biological sciences/Genetics"},{"id":64015087,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-03-24T08:26:58+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-06 11:38:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8982208","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8982208","identity":"rs-8982208","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}