APOBEC-Induced Mutagenesis in Cancer.

The initiation, progression, and relapse of cancers often result from mutations occurring within somatic cells. Consequently, processes that elevate mutation rates accelerate carcinogenesis and hinder the development of long-lasting therapeutics. Recent sequencing of human cancer genomes has identified patterns of mutations, termed mutation signatures, many of which correspond to specific environmentally induced and endogenous mutation processes. Some of the most frequently observed mutation signatures are caused by dysregulated activity of APOBECs, which deaminate cytidines in single-stranded DNA at specific sequence motifs causing C-to-T and C-to-G substitutions. In humans, APOBEC-generated genetic heterogeneity in tumor cells contributes to carcinogenesis, metastasis, and resistance to therapeutics. Here, we review the current understanding of APOBECs' role in cancer mutagenesis and impact on disease and the biological processes that influence APOBEC mutagenic capacity.

Her2 amplification, Rel-A, and Bach1 can influence APOBEC3A expression in breast cancer cells.

APOBEC-induced mutations occur in 50% of sequenced human tumors, with APOBEC3A (A3A) being a major contributor to mutagenesis in breast cancer cells. The mechanisms that cause A3A activation and mutagenesis in breast cancers are still unknown. Here, we describe factors that influence basal A3A mRNA transcript levels in breast cancer cells. We found that basal A3A mRNA correlates with A3A protein levels and predicts the amount of APOBEC signature mutations in a panel of breast cancer cell lines, indicating that increased basal transcription may be one mechanism leading to breast cancer mutagenesis. We also show that alteration of ERBB2 expression can drive A3A mRNA levels, suggesting the enrichment of the APOBEC mutation signature in Her2-enriched breast cancer could in part result from elevated A3A transcription. Hierarchical clustering of transcripts in primary breast cancers determined that A3A mRNA was co-expressed with other genes functioning in viral restriction and interferon responses. However, reduction of STAT signaling via inhibitors or shRNA in breast cancer cell lines had only minor impact on A3A abundance. Analysis of single cell RNA-seq from primary tumors indicated that A3A mRNA was highest in infiltrating immune cells within the tumor, indicating that correlations of A3A with STAT signaling in primary tumors may be result from higher immune infiltrates and are not reflective of STAT signaling controlling A3A expression in breast cancer cells. Analysis of ATAC-seq data in multiple breast cancer cell lines identified two transcription factor sites in the APOBEC3A promoter region that could promote A3A transcription. We determined that Rel-A, and Bach1, which have binding sites in these peaks, elevated basal A3A expression. Our findings highlight a complex and variable set of transcriptional activators for A3A in breast cancer cells.

Genetic inhibitors of APOBEC3B-induced mutagenesis.

The cytidine deaminases APOBEC3A (A3A) and APOBEC3B (A3B) are prominent mutators of human cancer genomes. However, tumor-specific genetic modulators of APOBEC-induced mutagenesis are poorly defined. Here, we used a screen to identify 61 gene deletions that increase A3B-induced mutations in yeast. We also determined whether each deletion was epistatic with Ung1 loss, which indicated whether the encoded factors participate in the homologous recombination (HR)-dependent bypass of A3B/Ung1-dependent abasic sites or suppress A3B-catalyzed deamination by protecting against aberrant formation of single-stranded DNA (ssDNA). We found that the mutation spectra of A3B-induced mutations revealed genotype-specific patterns of strand-specific ssDNA formation and nucleotide incorporation across APOBEC-induced lesions. Combining these three metrics, we were able to establish a multifactorial signature of APOBEC-induced mutations specific to (1) failure to remove H3K56 acetylation, (2) defective CTF18-RFC complex function, and (3) defective HR-mediated bypass of APOBEC-induced lesions. We extended these results by analyzing mutation data for human tumors and found BRCA1/2-deficient breast cancers display three- to fourfold more APOBEC-induced mutations. Mirroring our results in yeast, Rev1-mediated C-to-G substitutions are mainly responsible for increased APOBEC-signature mutations in BRCA1/2-deficient tumors, and these mutations associate with lagging strand synthesis during replication. These results identify important factors that influence DNA replication dynamics and likely the abundance of APOBEC-induced mutation during tumor progression. They also highlight a novel role for BRCA1/2 during HR-dependent lesion bypass of APOBEC-induced lesions during cancer cell replication.

Genome-wide maps of rare and atypical UV photoproducts reveal distinct patterns of damage formation and mutagenesis in yeast chromatin.

Ultraviolet (UV) light induces different classes of mutagenic photoproducts in DNA, namely cyclobutane pyrimidine dimers (CPDs), 6-4 photoproducts (6-4PPs), and atypical thymine-adenine photoproducts (TA-PPs). CPD formation is modulated by nucleosomes and transcription factors (TFs), which has important ramifications for Ultraviolet (UV) mutagenesis. How chromatin affects the formation of 6-4PPs and TA-PPs is unclear. Here, we use UV damage endonuclease-sequencing (UVDE-seq) to map these UV photoproducts across the yeast genome. Our results indicate that nucleosomes, the fundamental building block of chromatin, have opposing effects on photoproduct formation. Nucleosomes induce CPDs and 6-4PPs at outward rotational settings in nucleosomal DNA but suppress TA-PPs at these settings. Our data also indicate that DNA binding by different classes of yeast TFs causes lesion-specific hotspots of 6-4PPs or TA-PPs. For example, DNA binding by the TF Rap1 generally suppresses CPD and 6-4PP formation but induces a TA-PP hotspot. Finally, we show that 6-4PP formation is strongly induced at the binding sites of TATA-binding protein (TBP), which is correlated with higher mutation rates in UV-exposed yeast. These results indicate that the formation of 6-4PPs and TA-PPs is modulated by chromatin differently than CPDs and that this may have important implications for UV mutagenesis.

CTCF binding modulates UV damage formation to promote mutation hot spots in melanoma.

Somatic mutations in DNA-binding sites for CCCTC-binding factor (CTCF) are significantly elevated in many cancers. Prior analysis has suggested that elevated mutation rates at CTCF-binding sites in skin cancers are a consequence of the CTCF-cohesin complex inhibiting repair of UV damage. Here, we show that CTCF binding modulates the formation of UV damage to induce mutation hot spots. Analysis of genome-wide CPD-seq data in UV-irradiated human cells indicates that formation of UV-induced cyclobutane pyrimidine dimers (CPDs) is primarily suppressed by CTCF binding but elevated at specific locations within the CTCF motif. Locations of CPD hot spots in the CTCF-binding motif coincide with mutation hot spots in melanoma. A similar pattern of damage formation is observed at CTCF-binding sites in vitro, indicating that UV damage modulation is a direct consequence of CTCF binding. We show that CTCF interacts with binding sites containing UV damage and inhibits repair by a model repair enzyme in vitro. Structural analysis and molecular dynamic simulations reveal the molecular mechanism for how CTCF binding modulates CPD formation.

Environmental influence on the Atlantic salmon transcriptome and methylome during sea lice infestations.

The fish's immune response is affected by different factors, including a wide range of environmental conditions that can also disrupt or promote changes in the host-pathogen interactions. How environmental conditions modulate the salmon genome during parasitism is poorly understood here. This study aimed to explore the environmental influence on the Salmo salar transcriptome and methylome infected with the sea louse Caligus rogercresseyi. Atlantic salmon were experimentally infected with lice at two temperatures (8 and 16 °C) and salinity conditions (32 and 26PSU). Fish tissues were collected from the infected Atlantic salmon for reduced representation bisulfite sequencing (RRBS) and whole transcriptome sequencing (RNA-seq) analysis. The parasitic load was highly divergent in the evaluated environmental conditions, where the lowest lice abundance was observed in fish infected at 8 °C/26PSU. Notably, transcriptome profile differences were statistically associated with the number of alternative splicing events in fish exposed to low temperature/salinity conditions. Furthermore, the temperature significantly affected the methylation level, where high values of differential methylation regions were observed at 16 °C. Also, the association between expression levels of spliced transcripts and their methylation levels was determined, revealing significant correlations with Ferroptosis and TLR KEEG pathways. This study supports the relevance of the environmental conditions during host-parasite interactions in marine ecosystems. The discovery of alternative splicing transcripts associated with DMRs is also discussed as a novel player in fish biology.

Essential Roles for Polymerase θ-Mediated End Joining in the Repair of Chromosome Breaks.

DNA polymerase theta (Pol θ)-mediated end joining (TMEJ) has been implicated in the repair of chromosome breaks, but its cellular mechanism and role relative to canonical repair pathways are poorly understood. We show that it accounts for most repairs associated with microhomologies and is made efficient by coupling a microhomology search to removal of non-homologous tails and microhomology-primed synthesis across broken ends. In contrast to non-homologous end joining (NHEJ), TMEJ efficiently repairs end structures expected after aborted homology-directed repair (5' to 3' resected ends) or replication fork collapse. It typically does not compete with canonical repair pathways but, in NHEJ-deficient cells, is engaged more frequently and protects against translocation. Cell viability is also severely impaired upon combined deficiency in Pol θ and a factor that antagonizes end resection (Ku or 53BP1). TMEJ thus helps to sustain cell viability and genome stability by rescuing chromosome break repair when resection is misregulated or NHEJ is compromised.

Characterization of sequence contexts that favor alternative end joining at Cas9-induced double-strand breaks.

Alternative end joining (alt-EJ) mechanisms, such as polymerase theta-mediated end joining, are increasingly recognized as important contributors to inaccurate double-strand break repair. We previously proposed an alt-EJ model whereby short DNA repeats near a double-strand break anneal to form secondary structures that prime limited DNA synthesis. The nascent DNA then pairs with microhomologous sequences on the other break end. This synthesis-dependent microhomology-mediated end joining (SD-MMEJ) explains many of the alt-EJ repair products recovered following I-SceI nuclease cutting in Drosophila. However, sequence-specific factors that influence SD-MMEJ repair remain to be fully characterized. Here, we expand the utility of the SD-MMEJ model through computational analysis of repair products at Cas9-induced double-strand breaks for 1100 different sequence contexts. We find evidence at single nucleotide resolution for sequence characteristics that drive successful SD-MMEJ repair. These include optimal primer repeat length, distance of repeats from the break, flexibility of DNA sequence between primer repeats, and positioning of microhomology templates relative to preferred primer repeats. In addition, we show that DNA polymerase theta is necessary for most SD-MMEJ repair at Cas9 breaks. The analysis described here includes a computational pipeline that can be utilized to characterize preferred mechanisms of alt-EJ repair in any sequence context.

Conversations about race in Black and White US families: Before and after George Floyd's death.

Research has shown that Black parents are more likely than White parents to have conversations about race with their children, but few studies have directly compared the frequency and content of these conversations and how they change in response to national events. Here we examine such conversations in the United States before and after the killing of George Floyd. Black parents had conversations more often than White parents, and they had more frequent conversations post-Floyd. White parents remained mostly unchanged and, if anything, were less likely to talk about being White and more likely to send colorblind messages. Black parents were also more worried than White parents-both that their children would experience racial bias and that their children would perpetrate racial bias, a finding that held both pre- and post-Floyd. Thus, even in the midst of a national moment on race, White parents remained relatively silent and unconcerned about the topic.

The emotional and mental health impact of the murder of George Floyd on the US population.

On May 25, 2020, George Floyd, an unarmed Black American male, was killed by a White police officer. Footage of the murder was widely shared. We examined the psychological impact of Floyd's death using two population surveys that collected data before and after his death; one from Gallup (117,568 responses from n = 47,355) and one from the US Census (409,652 responses from n = 319,471). According to the Gallup data, in the week following Floyd's death, anger and sadness increased to unprecedented levels in the US population. During this period, more than a third of the US population reported these emotions. These increases were more pronounced for Black Americans, nearly half of whom reported these emotions. According to the US Census Household Pulse data, in the week following Floyd's death, depression and anxiety severity increased among Black Americans at significantly higher rates than that of White Americans. Our estimates suggest that this increase corresponds to an additional 900,000 Black Americans who would have screened positive for depression, associated with a burden of roughly 2.7 million to 6.3 million mentally unhealthy days.

Drinking as affective labour: A discussion of Australian men working in hospitality and corporate workplaces.

In the public imaginary, drinking is often thought of as a behaviour separate from individuals' formal labour practices, but studies increasingly highlight the complex ways alcohol is entwined with work. Building on recent conceptual developments in the sociological fields of youth, health and work, we illustrate how drinking can be productively understood as 'affective labour', and thus itself a form of work that generates valuable embodied states and atmospheres. To do so, we draw on data from six focus groups with men coworkers from three hospitality workplaces and three corporate workplaces in Victoria. For the corporate groups, work-related drinking was tied to an unravelling of certain professional affects and facilitated harmonious and productive workplace relationships, but also introduced risks ranging from embarrassment to sexual harassment. For hospitality workers, drinking was more deeply enmeshed in workplace relationships and, for one group, drinking on-shift was positively framed as creating an affect and atmosphere that appealed to clientele, despite taking a toll on workers' wellbeing. In both settings not drinking risked limiting one's ability to get on colleagues' affective 'level'. Our data deepens current understandings of how drinking cultures may be woven through occupational settings, produce value for organisations and introduce unique potential for exclusion.

Asymmetric repair of UV damage in nucleosomes imposes a DNA strand polarity on somatic mutations in skin cancer.

Nucleosomes inhibit excision repair of DNA damage caused by ultraviolet (UV) light, and it has been generally assumed that repair inhibition is equivalent on both sides of the nucleosome dyad. Here, we use genome-wide repair data to show that repair of UV damage in nucleosomes is asymmetric. In yeast, nucleosomes inhibit nucleotide excision repair (NER) of the nontranscribed strand (NTS) of genes in an asymmetric manner, with faster repair of UV damage occurring on the 5' side of the nucleosomal DNA. Analysis of genomic repair data from UV-irradiated human cells indicates that NER activity along the NTS is also elevated on the 5' side of nucleosomes, consistent with the repair asymmetry observed in yeast nucleosomes. Among intergenic nucleosomes, repair activity is elevated on the 5' side of both DNA strands. The distribution of somatic mutations in nucleosomes shows the opposite asymmetry in NER-proficient skin cancers, but not in NER-deficient cancers, indicating that asymmetric repair of nucleosomal DNA imposes a strand polarity on UV mutagenesis. Somatic mutations are enriched on the relatively slow-repairing 3' side of the nucleosomal DNA, particularly at positions where the DNA minor groove faces away from the histone octamer. Asymmetric repair and mutagenesis are likely caused by differential accessibility of the nucleosomal DNA, a consequence of its left-handed wrapping around the histone octamer.

Epigenetic-associated phenotypic plasticity of the ocean acidification-acclimated edible oyster in the mariculture environment.

For marine invertebrates with a pelagic-benthic life cycle, larval exposure to ocean acidification (OA) can affect adult performance in response to another environmental stressor. This carry-over effect has the potential to alter phenotypic traits. However, the molecular mechanisms that mediate "OA"-triggered carry-over effects have not been explored despite such information being key to improving species fitness and management strategies for aquafarming. This study integrated the genome-wide DNA methylome and transcriptome to examine epigenetic modification-mediated carry-over OA impacts on phenotypic traits of the ecologically and commercially important oyster species Crassostrea hongkongensis under field conditions. Larvae of C. hongkongensis were exposed to control pH 8.0 and low pH 7.4 conditions, mimicking near future OA scenario in their habitat, before being outplanted as post-metamorphic juveniles at two mariculture field sites with contrasting environmental stressors for 9 months. The larval carry-over OA effect was found to have persistent impacts on the growth and survival trade-off traits on the outplanted juveniles, although the beneficial or adverse effect depended on the environmental conditions at the outplanted sites. Site-specific plasticity was demonstrated with a diverse DNA methylation-associated gene expression profile, with signal transduction and the endocrine system being the most common and highly enriched functions. Highly methylated exons prevailed in the key genes related to general metabolic and endocytic responses and these genes are evolutionarily conserved in various marine invertebrates in response to OA. These results suggest that oysters with prior larval exposure history to OA had the ability to trigger rapid local adaptive responses via epigenetic modification to cope with multiple stressors in the field.

Triploid Pacific oysters exhibit stress response dysregulation and elevated mortality following heatwaves.

Polyploidy has been suggested to negatively impact environmental stress tolerance, resulting in increased susceptibility to extreme climate events. In this study, we compared the genomic and physiological response of diploid (2n) and triploid (3n) Pacific oysters (Crassostrea gigas) to conditions present during an atmospheric heatwave that impacted the Pacific Northwestern region of the United States in the summer of 2021. Climate stressors were applied either singly (single stressor; elevated seawater temperature, 30°C) or in succession (multiple stressor; elevated seawater temperature followed by aerial emersion at 44°C), replicating conditions present within the intertidal over a tidal cycle during the event. Oyster mortality rate was elevated within stress treatments with respect to the control and was significantly higher in triploids than diploids following multiple stress exposure (36.4% vs. 14.8%). Triploids within the multiple stressor treatment exhibited signs of energetic limitation, including metabolic depression, a significant reduction in ctenidium Na+ /K+ ATPase activity, and the dysregulated expression of genes associated with stress response, innate immunity, glucose metabolism, and mitochondrial function. Functional enrichment analysis of ploidy-specific gene sets identified that biological processes associated with metabolism, stress tolerance, and immune function were overrepresented within triploids across stress treatments. Our results suggest that triploidy impacts the transcriptional regulation of key processes that underly the stress response of Pacific oysters, resulting in downstream shifts in physiological tolerance limits that may increase susceptibility to extreme climate events that present multiple environmental stressors. The impact of chromosome set manipulation on the climate resilience of marine organisms has important implications for domestic food security within future climate scenarios, especially as triploidy induction becomes an increasingly popular tool to elicit reproductive control across a wide range of species used within marine aquaculture.

The effect of group status on children's hierarchy-reinforcing beliefs.

Members of advantaged groups are more likely than members of disadvantaged groups to think, feel, and behave in ways that reinforce their group's position within the hierarchy. This study examined how children's status within a group-based hierarchy shapes their beliefs about the hierarchy and the groups that comprise it in ways that reinforce the hierarchy. To do this, we randomly assigned children (4-8 years; N = 123; 75 female, 48 male; 21 Asian, 9 Black, 21 Latino/a, 1 Middle-Eastern/North-African, 14 multiracial, 41 White, 16 not-specified) to novel groups that differed in social status (advantaged, disadvantaged, neutral third-party) and assessed their beliefs about the hierarchy. Across five separate assessments, advantaged-group children were more likely to judge the hierarchy to be fair, generalizable, and wrong to challenge and were more likely to hold biased intergroup attitudes and exclude disadvantaged group members. In addition, with age, children in both the advantaged- and disadvantaged-groups became more likely to see membership in their own group as inherited, while at the same time expecting group-relevant behaviors to be determined more by the environment. With age, children also judged the hierarchy to be more unfair and expected the hierarchy to generalize across contexts. These findings provide novel insights into how children's position within hierarchies can contribute to the formation of hierarchy-reinforcing beliefs. RESEARCH HIGHLIGHTS: A total of 123 4-8-year-olds were assigned to advantaged, disadvantaged, and third-party groups within a hierarchy and were assessed on seven hierarchy-reinforcing beliefs about the hierarchy. Advantaged children were more likely to say the hierarchy was fair, generalizable, and wrong to challenge and to hold intergroup biases favoring advantaged group members. With age, advantaged- and disadvantaged-group children held more essentialist beliefs about membership in their own group, but not the behaviors associated with their group. Results suggest that advantaged group status can shape how children perceive and respond to the hierarchies they are embedded within.

Being from a highly resourced context predicts believing that others are highly resourced: An early developing worldview that stymies resource sharing.

We tested whether children's and adults' resource levels predicted their beliefs about resources (Study 1) and whether those beliefs shaped their willingness to share their resources with others (Study 2). In Study 1, we found that among adults (n = 230, 59.1% female, 72.6% White) and young children (n = 109, 4-6 years old, 56% female, 33% White), increased resource level predicted increases in the belief that others have lots of resources. In Study 2, we found that adults (n = 495, 52.5% female, 69.1% White) and young children (n = 154, 4-5 years old, 52.6% female, 36.4% White) randomly assigned to believe that others have lots of resources were less likely to share their own resources with others. Implications for reducing economic inequality are discussed.

Impact and evaluation of an online culinary nutrition course for health, education and industry professionals to promote vegetable knowledge and consumption.

BACKGROUND: Poor diet, including inadequate vegetable intake, is a leading risk factor for noncommunicable disease. Culinary and nutrition education provided to trainee and practising health and education professionals is an emerging strategy to promote improved dietary intake, including vegetable consumption. We evaluated the impact and feasibility of an online culinary medicine and nutrition (CM/CN) short course for health, education and vegetable industry professionals. The course aimed to improve participants' skills and confidence to prepare vegetables, knowledge of evidence-based nutrition information and recommendations for improving vegetable consumption and diet quality. METHODS: A pre-post study consisting of two separate groups participating in two course rounds recruited practising professionals (n = 30) working in health; community, adult and/or culinary education; and the vegetable industry. Evaluation assessed diet quality, vegetable consumption barriers, cooking and food skill confidence, nutrition knowledge and process measures. RESULTS: Seventeen participants (68%) completed the programme. Pre- to postintervention statistically significant increases in vegetables (M 1.3, SD 2.2), fruit (M 1.6, SD 3.1), and breads and cereal (M 1.1, SD 1.7) intakes were observed. Statistically significant increases and large effect sizes for mean food skill confidence scores (M 8.9, SD 15.4, Cohen's d 0.56) and nutrition knowledge scores (M 6.2, SD 15.4, Cohen's d 0.83) were also observed pre- to postintervention. CONCLUSIONS: The short online course was feasible and improved diet quality, food skill confidence and nutrition knowledge. Online CM/CN education for practising professionals represents a promising area of research. Future research involving a larger study sample and a more rigorous study design such as a randomised control trial is warranted.

Facilitators and barriers to providing culinary nutrition, culinary medicine and behaviour change support: An online cross-sectional survey of Australian health and education professionals.

BACKGROUND: An Australia wide cross-sectional online survey examined facilitators and barriers of health and education professionals to providing culinary nutrition (CN) and culinary medicine (CM) education and behaviour change support in usual practice, in addition to identifying continuing professional development (CPD) needs in this domain. METHODS: Survey items included socio-demographic characteristics, cooking and food skills confidence, nutrition knowledge (PKB-7), fruit and vegetable intake (FAVVA) and CPD needs. Data were summarised descriptively. RESULTS: Of 277 participants, 65% were likely/somewhat likely to participate in CN CPD. Mean (SD) cooking and food skill confidence scores were 73 (17.5) and 107.2 (24), out of 98 and 147, respectively. Mean PKB-7 score was 3.7 (1.4), out of 7. Mean FAVVA score was 98 (29), out of 190. CONCLUSIONS: Gaps in knowledge and limited time were the greatest modifiable barriers to providing CM/CN education and behaviour change support in practice. Health and education professionals are interested in CPD conducted by dietitians and culinary professionals to enhance their knowledge of CM/CN and behaviour change support.

Polymerase δ promotes chromosomal rearrangements and imprecise double-strand break repair.

Recent studies have implicated DNA polymerases θ (Pol θ) and ß (Pol ß) as mediators of alternative nonhomologous end-joining (Alt-NHEJ) events, including chromosomal translocations. Here we identify subunits of the replicative DNA polymerase δ (Pol δ) as promoters of Alt-NHEJ that results in more extensive intrachromosomal mutations at a single double-strand break (DSB) and more frequent translocations between two DSBs. Depletion of the Pol δ accessory subunit POLD2 destabilizes the complex, resulting in degradation of both POLD1 and POLD3 in human cells. POLD2 depletion markedly reduces the frequency of translocations with sequence modifications but does not affect the frequency of translocations with exact joins. Using separation-of-function mutants, we show that both the DNA synthesis and exonuclease activities of the POLD1 subunit contribute to translocations. As described in yeast and unlike Pol θ, Pol δ also promotes homology-directed repair. Codepletion of POLD2 with 53BP1 nearly eliminates translocations. POLD1 and POLD2 each colocalize with phosphorylated H2AX at ionizing radiation-induced DSBs but not with 53BP1. Codepletion of POLD2 with either ligase 3 (LIG3) or ligase 4 (LIG4) does not further reduce translocation frequency compared to POLD2 depletion alone. Together, these data support a model in which Pol δ promotes Alt-NHEJ in human cells at DSBs, including translocations.

Mechanistic basis for microhomology identification and genome scarring by polymerase theta.

DNA polymerase theta mediates an end joining pathway (TMEJ) that repairs chromosome breaks. It requires resection of broken ends to generate long, 3' single-stranded DNA tails, annealing of complementary sequence segments (microhomologies) in these tails, followed by microhomology-primed synthesis sufficient to resolve broken ends. The means by which microhomologies are identified is thus a critical step in this pathway, but is not understood. Here we show microhomologies are identified by a scanning mechanism initiated from the 3' terminus and favoring bidirectional progression into flanking DNA, typically to a maximum of 15 nucleotides into each flank. Polymerase theta is frequently insufficiently processive to complete repair of breaks in microhomology-poor, AT-rich regions. Aborted synthesis leads to one or more additional rounds of microhomology search, annealing, and synthesis; this promotes complete repair in part because earlier rounds of synthesis generate microhomologies de novo that are sufficiently long that synthesis is more processive. Aborted rounds of synthesis are evident in characteristic genomic scars as insertions of 3 to 30 bp of sequence that is identical to flanking DNA ("templated" insertions). Templated insertions are present at higher levels in breast cancer genomes from patients with germline BRCA1/2 mutations, consistent with an addiction to TMEJ in these cancers. Our work thus describes the mechanism for microhomology identification and shows how it both mitigates limitations implicit in the microhomology requirement and generates distinctive genomic scars associated with pathogenic genome instability.