APOBEC3G Is a p53-Dependent Restriction Factor in Respiratory Syncytial Virus Infection of Human Cells Included in the p53/Immune Axis.

Identifying and understanding genetic factors that influence the propagation of the human respiratory syncytial virus (RSV) can lead to health benefits and possibly augment recent vaccine approaches. We previously identified a p53/immune axis in which the tumor suppressor p53 directly regulates the expression of immune system genes, including the seven members of the APOBEC3 family of DNA cytidine deaminases (A3), which are innate immune sentinels against viral infections. Here, we examined the potential p53 and A3 influence in RSV infection, as well as the overall p53-dependent cellular and p53/immune axis responses to infection. Using a paired p53 model system of p53+ and p53- human lung tumor cells, we found that RSV infection activates p53, leading to the altered p53-dependent expression of A3D, A3F, and A3G, along with p53 site-specific binding. Focusing on A3G because of its 10-fold-greater p53 responsiveness to RSV, the overexpression of A3G can reduce RSV viral replication and syncytial formation. We also observed that RSV-infected cells undergo p53-dependent apoptosis. The study was expanded to globally address at the transcriptional level the p53/immune axis response to RSV. Nearly 100 genes can be directly targeted by the p53/immune axis during RSV infection based on our p53BAER analysis (Binding And Expression Resource). Overall, we identify A3G as a potential p53-responsive restriction factor in RSV infection. These findings have significant implications for RSV clinical and therapeutic studies and other p53-influenced viral infections, including using p53 adjuvants to boost the response of A3 genes.

Etoposide-induced DNA damage is increased in p53 mutants: identification of ATR and other genes that influence effects of p53 mutations on Top2-induced cytotoxicity.

The functional status of the tumor suppressor p53 is a critical component in determining the sensitivity of cancer cells to many chemotherapeutic agents. DNA topoisomerase II (Top2) plays essential roles in DNA metabolism and is the target of FDA approved chemotherapeutic agents. Topoisomerase targeting drugs convert the enzyme into a DNA damaging agent and p53 influences cellular responses to these agents. We assessed the impact of the loss of p53 function on the formation of DNA damage induced by the Top2 poison etoposide. Using human HCT116 cells, we found resistance to etoposide in cell growth assays upon the functional loss of p53. Nonetheless, cells lacking fully functional p53 were etoposide hypersensitive in clonogenic survival assays. This complex role of p53 led us to directly examine the effects of p53 status on topoisomerase-induced DNA damage. A deficiency in functional p53 resulted in elevated levels of the Top2 covalent complexes (Top2cc) in multiple cell lines. Employing genome-wide siRNA screens, we identified a set of genes for which reduced expression resulted in enhanced synthetic lethality upon etoposide treatment of p53 defective cells. We focused on one hit from this screen, ATR, and showed that decreased expression sensitized the p53-defective cells to etoposide in all assays and generated elevated levels of Top2cc in both p53 proficient and deficient cells. Our findings suggest that a combination of etoposide treatment with functional inactivation of DNA repair in p53 defective cells could be used to enhance the therapeutic efficacy of Top2 targeting agents.

p53-responsive TLR8 SNP enhances human innate immune response to respiratory syncytial virus.

The Toll-like receptor 8 (TLR8) has an important role in innate immune responses to RNA viral infections, including respiratory syncytial virus (RSV). We previously reported that TLR8 expression was increased directly by the tumor suppressor and transcription factor p53 via a single nucleotide polymorphism (SNP) (rs3761624) in the TLR8 promoter, thereby placing TLR8 in the p53/immune axis. Because this SNP is in linkage disequilibrium with other SNPs associated with several infectious diseases, we addressed the combined influence of p53 and the SNP on downstream inflammatory signaling in response to a TLR8 cognate ssRNA ligand. Using human primary lymphocytes, p53 induction by chemotherapeutic agents such as ionizing radiation caused SNP-dependent synergistic increases in IL-6 following incubation with an ssRNA ligand, as well as TLR8 RNA and protein expression along with p53 binding at the TLR-p53 SNP site. Because TLR8 is X-linked, the increases were generally reduced in heterozygous females. We found a corresponding association of the p53-responsive allele with RSV disease severity in infants hospitalized with RSV infection. We conclude that p53 can strongly influence TLR8-mediated immune responses and that knowledge of the p53-responsive SNP can inform diagnosis and prognosis of RSV disease and other diseases that might have a TLR8 component, including cancer.

Revealing a human p53 universe.

p53 transcriptional networks are well-characterized in many organisms. However, a global understanding of requirements for in vivo p53 interactions with DNA and relationships with transcription across human biological systems in response to various p53 activating situations remains limited. Using a common analysis pipeline, we analyzed 41 data sets from genome-wide ChIP-seq studies of which 16 have associated gene expression data, including our recent primary data with normal human lymphocytes. The resulting extensive analysis, accessible at p53 BAER hub via the UCSC browser, provides a robust platform to characterize p53 binding throughout the human genome including direct influence on gene expression and underlying mechanisms. We establish the impact of spacers and mismatches from consensus on p53 binding in vivo and propose that once bound, neither significantly influences the likelihood of expression. Our rigorous approach revealed a large p53 genome-wide cistrome composed of >900 genes directly targeted by p53. Importantly, we identify a core cistrome signature composed of genes appearing in over half the data sets, and we identify signatures that are treatment- or cell-specific, demonstrating new functions for p53 in cell biology. Our analysis reveals a broad homeostatic role for human p53 that is relevant to both basic and translational studies.

ETV7-Mediated DNAJC15 Repression Leads to Doxorubicin Resistance in Breast Cancer Cells.

Breast cancer treatment often includes Doxorubicin as adjuvant as well as neoadjuvant chemotherapy. Despite its cytotoxicity, cells can develop drug resistance to Doxorubicin. Uncovering pathways and mechanisms involved in drug resistance is an urgent and critical aim for breast cancer research oriented to improve treatment efficacy. Here we show that Doxorubicin and other chemotherapeutic drugs induce the expression of ETV7, a transcriptional repressor member of ETS family of transcription factors. The ETV7 expression led to DNAJC15 down-regulation, a co-chaperone protein whose low expression was previously associated with drug resistance in breast and ovarian cancer. There was a corresponding reduction in Doxorubicin sensitivity of MCF7 and MDA-MB-231 breast cancer cells. We identified the binding site for ETV7 within DNAJC15 promoter and we also found that DNA methylation may be a factor in ETV7-mediated DNAJC15 transcriptional repression. These findings of an inverse correlation between ETV7 and DNAJC15 expression in MCF7 cells in terms of Doxorubicin resistance, correlated well with treatment responses of breast cancer patients with recurrent disease, based on our analyses of reported genome-wide expression arrays. Moreover, we demonstrated that ETV7-mediated Doxorubicin-resistance involves increased Doxorubicin efflux via nuclear pumps, which could be rescued in part by DNAJC15 up-regulation. With this study, we propose a novel role for ETV7 in breast cancer, and we identify DNAJC15 as a new target gene responsible for ETV7-mediated Doxorubicin-resistance. A better understanding of the opposing impacts of Doxorubicin could improve the design of combinatorial adjuvant regimens with the aim of avoiding resistance and relapse.

The global role for Cdc13 and Yku70 in preventing telomere resection across the genome.

Yeast Cdc13 protein (related to human CTC1) maintains telomere stability by preventing 5'-3' end resection. While Cdc13 and Yku70/Yku80 proteins appear to prevent excessive resection, their combined contribution to maintenance of telomere ends across the genome and their relative roles at specific ends of different chromosomes have not been addressable because Cdc13 and Yku70/Yku80 double mutants are sickly. Using our PFGE-shift approach where large resected molecules have slower pulse field gel electrophoresis mobilities, along with methods for maintaining viable double mutants, we address end-resection on most chromosomes as well as telomere end differences. In this global approach to looking at ends of most chromosomes, we identify chromosomes with 1-end resections and end-preferences. We also identify chromosomes with resection at both ends, previously not possible. 10-20% of chromosomes exhibit PFGE-shift when cdc13-1 cells are switched to restrictive temperature (37 °C). In yku70Δ cdc13-1 mutants, there is a telomere resection "storm" with approximately half the chromosomes experiencing at least 1-end resection, ∼10 kb/telomere, due to exonuclease1 and many exhibiting 2-end resection. Unlike for random internal chromosome breaks, resection of telomere ends is not coordinated. Telomere restitution at permissive temperature is rapid (<1 h) in yku70Δ cdc13-1 cells. Surprisingly, survival can be high although strain background dependent. Given large amount of resected telomeres, we examined associated proteins. Up to 90% of cells have ≥1 Rfa1 (RPA) focus and 60% have multiple foci when ∼30-40 telomeres/cell are resected. The ends are dispersed in the nucleus suggesting wide distribution of resected telomeres across nuclear space. The previously reported Rad52 nuclear centers of repair for random DSBs also appear in cells with many resected telomere ends, suggesting a Rad52 commonality to the organization of single strand ends and/or limitation on interactions of single-strand ends with Rad52.

Increased LOH due to Defective Sister Chromatid Cohesion Is due Primarily to Chromosomal Aneuploidy and not Recombination.

Loss of heterozygosity (LOH) is an important factor in cancer, pathogenic fungi, and adaptation to changing environments. The sister chromatid cohesion process (SCC) suppresses aneuploidy and therefore whole chromosome LOH. SCC is also important to channel recombinational repair to sister chromatids, thereby preventing LOH mediated by allelic recombination. There is, however, insufficient information about the relative roles that the SCC pathway plays in the different modes of LOH. Here, we found that the cohesin mutation mcd1-1, and other mutations in SCC, differentially affect the various types of LOH. The greatest effect, by three orders of magnitude, was on whole chromosome loss (CL). In contrast, there was little increase in recombination-mediated LOH, even for telomeric markers. Some of the LOH events that were increased by SCC mutations were complex, i.e., they were the result of several chromosome transactions. Although these events were independent of POL32, the most parsimonious way to explain the formation of at least some of them was break-induced replication through the centromere. Interestingly, the mcd1-1 pol32Δ double mutant showed a significant reduction in the rate of CL in comparison with the mcd1-1 single mutant. Our results show that defects in SCC allow the formation of complex LOH events that, in turn, can promote drug or pesticide resistance in diploid microbes that are pathogenic to humans or plants.

Youth-Adult Connectedness:: A Key Protective Factor for Adolescent Health.

Over the past 30 years, prevention science in the adolescent health field has moved from interventions focused on preventing single problem behaviors to efforts employing a dual approach, addressing risk factors that predict problems while simultaneously nurturing protective factors and promoting positive development. Through an examination of previous research and empirical case examples with vulnerable youth, this article considers the hypothesis that adolescents' sense of connectedness to caring adults acts as a protective factor against a range of risk behaviors. Multivariate analyses with existing data examined indicators of youth-adult connectedness among two groups at high risk for poor health outcomes: (1) mentor-youth relationship quality in an urban, ethnically diverse sample of students in a school-based mentoring program (2014 survey, N=239); and (2) parent-youth connectedness in a statewide sample of high school students who reported homelessness in the past year (2013 survey, N=3,627). For youth in the mentoring program, a high-quality youth-mentor relationship was significantly associated with positive social, academic, and health-related behaviors. Among students who experienced homelessness, all measures of parent connectedness were significantly associated with lower sexual risk levels. Collectively, findings from these analyses and previously published studies by this research group provide evidence that strong, positive relationships with parents and other caring adults protect adolescents from a range of poor health-related outcomes and promote positive development. Youth-adult connectedness appears to be foundational for adolescent health and well-being. Program, practice, and policy decisions should consider what strengthens or hinders caring, connected youth-adult relationships.

The Cytidine Deaminase APOBEC3 Family Is Subject to Transcriptional Regulation by p53.

The APOBEC3 (A3) family of proteins are DNA cytidine deaminases that act as sentinels in the innate immune response against retroviral infections and are responsive to IFN. Recently, a few A3 genes were identified as potent enzymatic sources of mutations in several human cancers. Using human cancer cells and lymphocytes, we show that under stress conditions and immune challenges, all A3 genes are direct transcriptional targets of the tumor suppressor p53. Although the expression of most A3 genes (including A3C and A3H) was stimulated by the activation of p53, treatment with the DNA-damaging agent doxorubicin or the p53 stabilizer Nutlin led to repression of the A3B gene. Furthermore, p53 could enhance IFN type-I induction of A3 genes. Interestingly, overexpression of a group of tumor-associated p53 mutants in TP53-null cancer cells promoted A3B expression. These findings establish that the "guardian of the genome" role ascribed to p53 also extends to a unique component of the immune system, the A3 genes, thereby integrating human immune and chromosomal stress responses into an A3/p53 immune axis.Implications: Activated p53 can integrate chromosomal stresses and immune responses through its influence on expression of APOBEC3 genes, which are key components of the innate immune system that also influence genomic stability. Mol Cancer Res; 15(6); 735-44. ©2017 AACR.

The novel p53 target TNFAIP8 variant 2 is increased in cancer and offsets p53-dependent tumor suppression.

Tumor necrosis factor-α-induced protein 8 (TNFAIP8) is a stress-response gene that has been associated with cancer, but no studies have differentiated among or defined the regulation or function of any of its several recently described expression variants. We found that TNFAIP8 variant 2 (v2) is overexpressed in multiple human cancers, whereas other variants are commonly downregulated in cancer (v1) or minimally expressed in cancer or normal tissue (v3-v6). Silencing v2 in cancer cells induces p53-independent inhibition of DNA synthesis, widespread binding of p53, and induction of target genes and p53-dependent cell cycle arrest and DNA damage sensitization. Cell cycle arrest induced by v2 silencing requires p53-dependent induction of p21. In response to the chemotherapeutic agent doxorubicin, p53 regulates v2 through binding to an intragenic enhancer, together indicating that p53 and v2 engage in complex reciprocal regulation. We propose that TNFAIP8 v2 promotes human cancer by broadly repressing p53 function, in essence offsetting p53-dependent tumor suppression.

Ligand dependent restoration of human TLR3 signaling and death in p53 mutant cells.

Diversity within the p53 transcriptional network can arise from a matrix of changes that include target response element sequences and p53 expression level variations. We previously found that wild type p53 (WT p53) can regulate expression of most innate immune-related Toll-like-receptor genes (TLRs) in human cells, thereby affecting immune responses. Since many tumor-associated p53 mutants exhibit change-of-spectrum transactivation from various p53 targets, we examined the ability of twenty-five p53 mutants to activate endogenous expression of the TLR gene family in p53 null human cancer cell lines following transfection with p53 mutant expression vectors. While many mutants retained the ability to drive TLR expression at WT levels, others exhibited null, limited, or change-of-spectrum transactivation of TLR genes. Using TLR3 signaling as a model, we show that some cancer-associated p53 mutants amplify cytokine, chemokine and apoptotic responses after stimulation by the cognate ligand poly(I:C). Furthermore, restoration of WT p53 activity for loss-of-function p53 mutants by the p53 reactivating drug RITA restored p53 regulation of TLR3 gene expression and enhanced DNA damage-induced apoptosis via TLR3 signaling. Overall, our findings have many implications for understanding the impact of WT and mutant p53 in immunological responses and cancer therapy.

The Shu complex promotes error-free tolerance of alkylation-induced base excision repair products.

Here, we investigate the role of the budding yeast Shu complex in promoting homologous recombination (HR) upon replication fork damage. We recently found that the Shu complex stimulates Rad51 filament formation during HR through its physical interactions with Rad55-Rad57. Unlike other HR factors, Shu complex mutants are primarily sensitive to replicative stress caused by MMS and not to more direct DNA breaks. Here, we uncover a novel role for the Shu complex in the repair of specific MMS-induced DNA lesions and elucidate the interplay between HR and translesion DNA synthesis. We find that the Shu complex promotes high-fidelity bypass of MMS-induced alkylation damage, such as N3-methyladenine, as well as bypassing the abasic sites generated after Mag1 removes N3-methyladenine lesions. Furthermore, we find that the Shu complex responds to ssDNA breaks generated in cells lacking the abasic site endonucleases. At each lesion, the Shu complex promotes Rad51-dependent HR as the primary repair/tolerance mechanism over error-prone translesion DNA polymerases. Together, our work demonstrates that the Shu complex's promotion of Rad51 pre-synaptic filaments is critical for high-fidelity bypass of multiple replication-blocking lesion.

Intimate partner violence in late adolescence and young adulthood and subsequent cardiovascular risk in adulthood.

BACKGROUND: Childhood maltreatment has been linked to adulthood cardiovascular disease (CVD). Little is known about the relationship between intimate partner violence (IPV) in late adolescence and young adulthood and CVD risk later in adulthood. PURPOSE: To examine whether IPV perpetration and victimization experienced in late adolescence and young adulthood are associated with CVD risk among adults in the United States and whether this relationship differs by sex. METHODS: Data include 9976 participants (50% female) in the National Longitudinal Study of Adolescent to Adult Health. Physical and sexual IPV were measured at wave 3 (2001/02) with items from the revised Conflict Tactics Scales. Participants'30-year risk of CVD was calculated at wave 4 (2008/09) using a Framingham prediction model. Linear regression models adjusted for confounders and IPV by sex interaction terms were tested to examine the relationship. RESULTS: The mean CVD risk score was 13.18% (95% CI: 12.71, 13.64). Aone-standard deviation increase in the victimization score was associated with a 0.28% (95% CI: 0.03, 0.54) increase in CVD risk. Perpetration was similarly positively associated with CVD risk (beta: 0.33, 95% CI: 0.03, 0.62). When measured as a composite, all violence types were associated with increased CVD risk but only prior exposure to both victimization and perpetration reached statistical significance (0.62%, 95% CI: 0.01, 1.22). No differences by sex were detected. CONCLUSIONS: Effect sizes are not large, but early detection of increased CVD risk in this relatively young population is notable and worthy of further study to inform the clinical response.

Recombinational repair of radiation-induced double-strand breaks occurs in the absence of extensive resection.

Recombinational repair provides accurate chromosomal restitution after double-strand break (DSB) induction. While all DSB recombination repair models include 5'-3' resection, there are no studies that directly assess the resection needed for repair between sister chromatids in G-2 arrested cells of random, radiation-induced 'dirty' DSBs. Using our Pulse Field Gel Electrophoresis-shift approach, we determined resection at IR-DSBs in WT and mutants lacking exonuclease1 or Sgs1 helicase. Lack of either reduced resection length by half, without decreased DSB repair or survival. In the exo1Δ sgs1Δ double mutant, resection was barely detectable, yet it only took an additional hour to achieve a level of repair comparable to WT and there was only a 2-fold dose-modifying effect on survival. Results with a Dnl4 deletion strain showed that remaining repair was not due to endjoining. Thus, similar to what has been shown for a single, clean HO-induced DSB, a severe reduction in resection tract length has only a modest effect on repair of multiple, dirty DSBs in G2-arrested cells. Significantly, this study provides the first opportunity to directly relate resection length at DSBs to the capability for global recombination repair between sister chromatids.

Quantitative Analysis of NF-κB Transactivation Specificity Using a Yeast-Based Functional Assay.

The NF-κB transcription factor family plays a central role in innate immunity and inflammation processes and is frequently dysregulated in cancer. We developed an NF-κB functional assay in yeast to investigate the following issues: transactivation specificity of NF-κB proteins acting as homodimers or heterodimers; correlation between transactivation capacity and in vitro DNA binding measurements; impact of co-expressed interacting proteins or of small molecule inhibitors on NF-κB-dependent transactivation. Full-length p65 and p50 cDNAs were cloned into centromeric expression vectors under inducible GAL1 promoter in order to vary their expression levels. Since p50 lacks a transactivation domain (TAD), a chimeric construct containing the TAD derived from p65 was also generated (p50TAD) to address its binding and transactivation potential. The p50TAD and p65 had distinct transactivation specificities towards seventeen different κB response elements (κB-REs) where single nucleotide changes could greatly impact transactivation. For four κB-REs, results in yeast were predictive of transactivation potential measured in the human MCF7 cell lines treated with the NF-κB activator TNFα. Transactivation results in yeast correlated only partially with in vitro measured DNA binding affinities, suggesting that features other than strength of interaction with naked DNA affect transactivation, although factors such as chromatin context are kept constant in our isogenic yeast assay. The small molecules BAY11-7082 and ethyl-pyruvate as well as expressed IkBα protein acted as NF-κB inhibitors in yeast, more strongly towards p65. Thus, the yeast-based system can recapitulate NF-κB features found in human cells, thereby providing opportunities to address various NF-κB functions, interactions and chemical modulators.

p53 amplifies Toll-like receptor 5 response in human primary and cancer cells through interaction with multiple signal transduction pathways.

The p53 tumor suppressor regulates transcription of genes associated with diverse cellular functions including apoptosis, growth arrest, DNA repair and differentiation. Recently, we established that p53 can modulate expression of Toll-like receptor (TLR) innate immunity genes but the degree of cross-talk between p53 and TLR pathways remained unclear. Here, using gene expression profiling we characterize the global effect of p53 on the TLR5-mediated transcription in MCF7 cells. We found that combined activation of p53 and TLR5 pathways synergistically increases expression of over 200 genes, mostly associated with immunity and inflammation. The synergy was observed in several human cancer cells and primary lymphocytes. The p53-dependent amplification of transcriptional response to TLR5 activation required expression of NFκB subunit p65 and was mediated by several molecular mechanisms including increased phosphorylation of p38 MAP kinase, PI3K and STAT3 signaling. Additionally, p53 induction increased cytokine expression in response to TNFα, another activator of NFκB and MAP kinase pathways, suggesting a broad interaction between p53 and these signaling pathways. The expression of many synergistically induced genes is elevated in breast cancer patients responsive to chemotherapy. We suggest that p53's capacity to enhance immune response could be exploited to increase antitumor immunity and to improve cancer treatment.

Bioinspired hybrid materials from spray-formed ceramic templates.

Thermally sprayed ceramics, when infiltrated with polymer, exhibit synergistic increases in strength and toughness. The structure of such composites-a dense, brick-mortar arrangement-is strikingly similar to that of nacre, as are the mechanisms underlying the robust mechanical behavior. This industrial-scale process thus presents an exciting tool for bio-mimetic exploration.

Tetrameric Ctp1 coordinates DNA binding and DNA bridging in DNA double-strand-break repair.

Ctp1 (also known as CtIP or Sae2) collaborates with Mre11-Rad50-Nbs1 to initiate repair of DNA double-strand breaks (DSBs), but its functions remain enigmatic. We report that tetrameric Schizosaccharomyces pombe Ctp1 contains multivalent DNA-binding and DNA-bridging activities. Through structural and biophysical analyses of the Ctp1 tetramer, we define the salient features of Ctp1 architecture: an N-terminal interlocking tetrameric helical dimer-of-dimers (THDD) domain and a central intrinsically disordered region (IDR) linked to C-terminal 'RHR' DNA-interaction motifs. The THDD, IDR and RHR are required for Ctp1 DNA-bridging activity in vitro, and both the THDD and RHR are required for efficient DSB repair in S. pombe. Our results establish non-nucleolytic roles of Ctp1 in binding and coordination of DSB-repair intermediates and suggest that ablation of human CtIP DNA binding by truncating mutations underlie the CtIP-linked Seckel and Jawad syndromes.

Leadership in adolescent health: developing the next generation of maternal child health leaders through mentorship.

Leadership development is a core value of Maternal Child Health Bureau training programs. Mentorship, an MCH Leadership Competency, has been shown to positively affect career advancement and research productivity. Improving mentorship opportunities for junior faculty and trainees may increase pursuit of careers in areas such as adolescent health research and facilitate the development of new leaders in the field. Using a framework of Developmental Networks, a group of MCH Leadership Education in Adolescent Health training program faculty developed a pilot mentoring program offered at the Society for Adolescent Health and Medicine Annual Meeting (2011-2013). The program matched ten interdisciplinary adolescent health fellows and junior faculty with senior mentors at other institutions with expertise in the mentee's content area of study in 2011. Participants were surveyed over 2 years. Respondents indicated they were "very satisfied" with their mentor match, and all agreed or strongly agreed that the mentoring process in the session was helpful, and that the mentoring relationships resulted in several ongoing collaborations and expanded their Developmental Networks. These results demonstrate that MCH programs can apply innovative strategies to disseminate the MCH Leadership Competencies to groups beyond MCH-funded training programs through programs at scientific meetings. Such innovations may enhance the structure of mentoring, further the development of new leaders in the field, and expand developmental networks to provide support for MCH professionals transitioning to leadership roles.

Break-induced replication is a source of mutation clusters underlying kataegis.

Clusters of simultaneous multiple mutations can be a source of rapid change during carcinogenesis and evolution. Such mutation clusters have been recently shown to originate from DNA damage within long single-stranded DNA (ssDNA) formed at resected double-strand breaks and dysfunctional replication forks. Here, we identify double-strand break (DSB)-induced replication (BIR) as another powerful source of mutation clusters that formed in nearly half of wild-type yeast cells undergoing BIR in the presence of alkylating damage. Clustered mutations were primarily formed along the track of DNA synthesis and were frequently associated with additional breakage and rearrangements. Moreover, the base specificity, strand coordination, and strand bias of the mutation spectrum were consistent with mutations arising from damage in persistent ssDNA stretches within unconventional replication intermediates. Altogether, these features closely resemble kataegic events in cancers, suggesting that replication intermediates during BIR may be the most prominent source of mutation clusters across species.