True Grit: Passion and persistence make an innovative course design work.

Our first two experiments on adapting a high-structure course model to an essentially open-enrollment university produced negative or null results. Our third experiment, however, proved more successful: performance improved for all students, and a large achievement gap that impacted underrepresented minority students under traditional lecturing closed. Although the successful design included preclass preparation videos, intensive active learning in class, and weekly practice exams, student self-report data indicated that total study time decreased. Faculty who have the grit to experiment and persevere in making evidence-driven changes to their teaching can reduce the inequalities induced by economic and educational disadvantage.

Identification of a New Antimicrobial, Desertomycin H, Utilizing a Modified Crowded Plate Technique.

The antibiotic-resistant bacteria-associated infections are a major global healthcare threat. New classes of antimicrobial compounds are urgently needed as the frequency of infections caused by multidrug-resistant microbes continues to rise. Recent metagenomic data have demonstrated that there is still biosynthetic potential encoded in but transcriptionally silent in cultivatable bacterial genomes. However, the culture conditions required to identify and express silent biosynthetic gene clusters that yield natural products with antimicrobial activity are largely unknown. Here, we describe a new antibiotic discovery scheme, dubbed the modified crowded plate technique (mCPT), that utilizes complex microbial interactions to elicit antimicrobial production from otherwise silent biosynthetic gene clusters. Using the mCPT as part of the antibiotic crowdsourcing educational program Tiny EarthTM, we isolated over 1400 antibiotic-producing microbes, including 62 showing activity against multidrug-resistant pathogens. The natural product extracts generated from six microbial isolates showed potent activity against vancomycin-intermediate resistant Staphylococcus aureus. We utilized a targeted approach that coupled mass spectrometry data with bioactivity, yielding a new macrolactone class of metabolite, desertomycin H. In this study, we successfully demonstrate a concept that significantly increased our ability to quickly and efficiently identify microbes capable of the silent antibiotic production.

Fragile site instability in Saccharomyces cerevisiae causes loss of heterozygosity by mitotic crossovers and break-induced replication.

Loss of heterozygosity (LOH) at tumor suppressor loci is a major contributor to cancer initiation and progression. Both deletions and mitotic recombination can lead to LOH. Certain chromosomal loci known as common fragile sites are susceptible to DNA lesions under replication stress, and replication stress is prevalent in early stage tumor cells. There is extensive evidence for deletions stimulated by common fragile sites in tumors, but the role of fragile sites in stimulating mitotic recombination that causes LOH is unknown. Here, we have used the yeast model system to study the relationship between fragile site instability and mitotic recombination that results in LOH. A naturally occurring fragile site, FS2, exists on the right arm of yeast chromosome III, and we have analyzed LOH on this chromosome. We report that the frequency of spontaneous mitotic BIR events resulting in LOH on the right arm of yeast chromosome III is higher than expected, and that replication stress by low levels of polymerase alpha increases mitotic recombination 12-fold. Using single-nucleotide polymorphisms between the two chromosome III homologs, we mapped the locations of recombination events and determined that FS2 is a strong hotspot for both mitotic reciprocal crossovers and break-induced replication events under conditions of replication stress.

The transitioning from trials to extended follow-up studies.

BACKGROUND: Investigators may elect to extend follow-up of participants enrolled in a randomized clinical trial after the trial comes to its planned end. The additional follow-up may be initiated to learn about longer term effects of treatments, including adverse events, costs related to treatment, or for reasons unrelated to treatment such as to observe the natural course of the disease using the established cohort from the trial. PURPOSE: We examine transitioning from trials to extended follow-up studies when the goal of additional follow-up is to observe longer term treatment effects. METHODS: We conducted a literature search in selected journals from 2000 to 2012 to identify trials that extended follow-up for the purpose of studying longer term treatment effects and extracted information on the operational and logistical issues in the transition. We also draw experience from three trials coordinated by the Johns Hopkins Coordinating Centers that made transitions to extended follow-up: the Alzheimer's Disease Anti-inflammatory Prevention Trial, Multicenter Uveitis Steroid Treatment trial, and Childhood Asthma Management Program. RESULTS: Transitions are not uncommon in multicenter clinical trials, even in trials that continued to the planned end of the trial. Transitioning usually necessitates new participant consents. If study infrastructure is not maintained during the transition, participants will be lost and re-establishing the staff and facilities will be costly. Merging data from the trial and follow-up study can be complicated by changes in data collection measures and schedules. LIMITATIONS: Our discussion and recommendations are limited to issues that we have experienced in transitions from trials to follow-up studies. DISCUSSION: We discuss issues such as maintaining funding, institutional review board and consent requirements, contacting participants, and combining data from the trial and follow-up phases. We conclude with a list of recommendations to facilitate transitions from a trial to an extended follow-up study.

A CURE Lab in Introductory Biology at a Regional Comprehensive University Negatively Impacts Student Success in the Associated Lecture Course Among Students from Groups Underrepresented in Science.

Course-based undergraduate research experiences (CUREs) have been proposed as a mechanism to democratize access to the benefits of apprentice-style scientific research to a broader diversity of students, promoting inclusivity and increasing student success and retention. As we evaluate CUREs, it is essential to explore their effectiveness within the environments of regional comprehensive universities and community colleges, because they are important access points for a wide variety of students. It is also important to address the potential influence of volunteer bias, where students can opt to enroll in either the CURE or a traditional lab, on the outcomes of CUREs. We evaluated a CURE at a regional comprehensive university under conditions both with and without volunteer bias. We find that nonvolunteer students report a lower sense of discovery and relevance of the CURE compared with students who volunteered for the course. Importantly, we also find that our replacement of the traditional lab class with a CURE resulted in lower scores on exams in the associated lecture course among students who are both BIPOC and Pell eligible. We call for additional research on the effects of CUREs at nonresearch-intensive institutions and without volunteer bias, to better understand the impact of these classes.

Daily trajectories of evening recovery experiences and their role for next-day mood.

Focusing on the definition of recovery as a process, we examined how the four core recovery experiences (i.e., psychological detachment, relaxation, control, and mastery) develop during the evening. We tested whether the specific developments of recovery experiences are important for next-day favorable mood states-beyond the mean levels of recovery experiences. We collected data from 92 employees who completed daily morning and afternoon surveys over 10 workdays. In the morning surveys, we implemented the day-reconstruction method to assess detailed information about employees' recovery experiences during several episodes of the previous evening. Our final data set included 477 morning surveys with a total of 1,998 episodes and 383 afternoon surveys. Multilevel growth curve analyses showed that, in general, psychological detachment, relaxation, and control follow a positive linear trend and mastery a negative quadratic trend during the evening. Moreover, path analyses showed that the day-level increase of psychological detachment is important for next-day mood. Specifically, we found that after evenings during which employees experienced a higher increase in psychological detachment than they usually did, they had higher favorable mood states in the subsequent afternoon. Further, our results did not support associations between day-level slopes of relaxation, control, and mastery as well as next-day mood. Hence, our study demonstrates that recovery experiences systematically change during an evening and that this change is partially relevant for next-afternoon mood. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

It's a match: The relevance of matching chronotypes for dual-earner couples' daily recovery from work.

Cohabiting dual-earner couples are increasingly common. However, previous recovery research mainly focused on employees independently of others, thereby overlooking an essential part of their life. Therefore, we take a closer look at dual-earner couples' recovery processes and link this research to a circadian perspective. We assumed that unfinished tasks impede engagement in time with the partner (absorption in joint activities, directing attention toward the partner) as well as recovery experiences (detachment, relaxation), whereas engagement in time with the partner should boost recovery experiences. Integrating a circadian perspective, we proposed that employees from couples with matching circadian preferences (chronotype) benefit more from engagement in time with their partner (i.e., stronger relationships with recovery experiences). Additionally, we explored whether a match between partners' chronotypes buffers the negative relationship between unfinished tasks and engagement in joint time. We conducted a daily diary study with 143 employees from 79 dual-earner couples, providing data on 1,052 days. A three-level path model showed that unfinished tasks were negatively related to absorption in joint activities and detachment, whereas absorption positively predicted recovery experiences. Furthermore, the couples' chronotype match mattered in the interplay with engagement in joint time: for couples with higher (vs. lower) chronotype match, experiencing detachment depended on absorption while for couples with lower (vs. higher) chronotype match, attention was even harmful for experiencing relaxation. Thus, it is crucial to consider employees' partners when investigating their recovery processes because employees cannot act independently if they also need to take their partner's circadian rhythms into account. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

The role of recovery for morning cognitive appraisal of work demands: A diary study.

We examined the role of daily recovery for morning cognitive appraisal of work demands in a daily diary study. We predicted that psychological detachment from work during the evening would be associated with changes in cognitive appraisal from afternoon to the next morning. Additionally, we predicted that these associations are mediated by state of being recovered in the morning. We collected data from 183 employees with 3 daily measurement occasions over 2 consecutive workweeks. We analyzed the data using multilevel path modeling with latent variance decomposition into within- and between-person variance parts. Results showed that psychological detachment predicted a decrease in hindrance and threat appraisal but no change in challenge appraisal from afternoon to morning. State of being recovered mediated the relationship between psychological detachment and threat appraisal but not hindrance appraisal. Psychological detachment was indirectly related to an increase in challenge appraisal via state of being recovered in the morning. Our results provide insights on predictors of cognitive appraisal and the role of recovery for cognitive processes in the stress process. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Low levels of DNA polymerase alpha induce mitotic and meiotic instability in the ribosomal DNA gene cluster of Saccharomyces cerevisiae.

The ribosomal DNA (rDNA) genes of Saccharomyces cerevisiae are located in a tandem array of about 150 repeats. Using a diploid with markers flanking and within the rDNA array, we showed that low levels of DNA polymerase alpha elevate recombination between both homologues and sister chromatids, about five-fold in mitotic cells and 30-fold in meiotic cells. This stimulation is independent of Fob1p, a protein required for the programmed replication fork block (RFB) in the rDNA. We observed that the fob1 mutation alone significantly increased meiotic, but not mitotic, rDNA recombination, suggesting a meiosis-specific role for this protein. We found that meiotic cells with low polymerase alpha had decreased Sir2p binding and increased Spo11p-catalyzed double-strand DNA breaks in the rDNA. Furthermore, meiotic crossover interference in the rDNA is absent. These results suggest that the hyper-Rec phenotypes resulting from low levels of DNA polymerase alpha in mitosis and meiosis reflect two fundamentally different mechanisms: the increased mitotic recombination is likely due to increased double-strand DNA breaks (DSBs) resulting from Fob1p-independent stalled replication forks, whereas the hyper-Rec meiotic phenotype results from increased levels of Spo11-catalyzed DSBs in the rDNA.

Noncanonical outcomes of break-induced replication produce complex, extremely long-tract gene conversion events in yeast.

Long-tract gene conversions (LTGC) can result from the repair of collapsed replication forks, and several mechanisms have been proposed to explain how the repair process produces this outcome. We studied LTGC events produced from repair collapsed forks at yeast fragile site FS2. Our analysis included chromosome sizing by contour-clamped homogeneous electric field electrophoresis, next-generation whole-genome sequencing, and Sanger sequencing across repair event junctions. We compared the sequence and structure of LTGC events in our cells to the expected qualities of LTGC events generated by proposed mechanisms. Our evidence indicates that some LTGC events arise from half-crossover during BIR, some LTGC events arise from gap repair, and some LTGC events can be explained by either gap repair or "late" template switch during BIR. Also based on our data, we propose that models of collapsed replication forks be revised to show not a one-end double-strand break (DSB), but rather a two-end DSB in which the ends are separated in time and subject to gap repair.

Natural vs. programmed cycles for frozen embryo transfer: study protocol for an investigator-initiated, randomized, controlled, multicenter clinical trial.

BACKGROUND: Randomized trials of assisted reproductive technology (ART) have been designed for outcomes of clinical pregnancy or live birth and have not been powered for obstetric outcomes such as preeclampsia, critical for maternal and fetal health. ART increasingly involves frozen embryo transfer (FET). Although there are advantages of FET, multiple studies have shown that risk of preeclampsia is increased with FET compared with fresh embryo transfer, and the reason for this difference is not clear. NatPro will compare the proportion of preeclampsia between two commonly used protocols for FET,modified natural and programmed cycle. METHODS: In this two-arm, parallel-group, multi-center randomized trial, NatPro will randomize 788 women to either modified natural or programmed FET and follow them for up to three FET cycles. Primary outcome will be the proportion of preeclampsia in women with a viable pregnancy assigned to a modified natural cycle FET (corpus luteum present) protocol compared to the proportion of preeclampsia in pregnant women assigned to a programmed FET (corpus luteum absent) protocol. Secondary outcomes will compare the proportion of live births and the proportion of preeclampsia with severe features between the protocols. CONCLUSION: This study has a potential significant impact on millions of women who pursue ART to build their families. NatPro is designed to provide clinically relevant guidance to inform patients and clinicians regarding maternal risk with programmed and modified natural cycle FET protocols. This study will also provide accurate point estimates regarding the likelihood of live birth with programmed and modified natural cycle FET. TRIAL REGISTRATION: ClinicalTrials.gov NCT04551807 . Registered on September 16, 2020.

Association of mild cognitive impairment and characteristic of COPD and overall health status in a cohort study.

Introduction: We evaluated risk factors and demographic characteristics of associated with mild cognitive impairment (MCI) in patients with COPD. Methods: 220 individuals with COPD enrolled in a cohort study designed to evaluate anxiety conducted at 16 clinical centers. Cognitive impairment was assessed with the Montreal Cognitive Assessment (MoCA), a cutoff score of <26 defined as MCI. Data were collected including spirometry, 6-minute walk test, symptom burden by COPD Assessment Test and dyspnea by Modified Medical Research Council, anxiety measured by Anxiety Inventory of Respiratory Disease, Generalized Anxiety Disorder-7 and Hospital Anxiety Depression Scale, depression by Patient Health Questionnaire-9 and health status by Patient Reported Outcomes Measurement Information System and sleep quality by the Pittsburg Sleep Quality Index. Results: The median age was 65 years and 54% of participants were male. 119(54%) of participants had MCI as classified by MoCA. In multivariable logistic regression, higher odds ratios (OR) (95% confidence interval) for MCI (MoCA) <26 were associated with increased years of age, 1.06 (1.02 -1-09, p<0.003); African-American race, 3.68(1.67-8.11, p<0.001); persistent phlegm, 2 (1.12-3.57, p<0.01) and sleep disturbance, 1.04(1.01-1.08, p<0.01). Conclusions: COPD patients commonly screen positive for MCI. Characteristics associated with MCI included age, African-American race, sleep disturbance and persistent phlegm.

Outperforming yet undervalued: Undergraduate women in STEM.

In spite of efforts to increase gender diversity across many science fields, women continue to encounter beliefs that they lack ability and talent. Undergraduate education is a critical time when peer influence may alter choice of majors and careers for women interested in science. Even in life science courses, in which women outnumber men, gender biases that emerge in peer-to-peer interactions during coursework may detract from women's interest and progress. This is the first study of which we are aware to document that women are outperforming men in both physical and life science undergraduate courses at the same institution, while simultaneously continuing to be perceived as less-able students. This is problematic because undergraduate women may not be able to escape gender-ability stereotypes even when they are outperforming men, which has important implications for 1) the recognition of women's achievements among their peers in undergraduate education and 2) retention of women in STEM disciplines and careers.

Patterns of positive and negative work reflection during leisure time: A latent profile analysis.

This study examined positive and negative work reflection during leisure time from a person-centered perspective using latent profile analysis. First, we examined whether quantitatively and qualitatively different work reflection profiles exist. Second, we investigated whether persons with different work reflection profiles differ in energetic well-being (i.e., exhaustion and vigor). We collected data from 1,036 young employees who answered 3 surveys with a time lag of 3 months each. We established the profile solution at the first measurement point and tested for differences in well-being at the second and third measurement point. We identified 6 work reflection profiles with 2 profiles displaying unbalanced levels of positive and negative work reflection (positive reflectors and negative reflectors) and 4 profiles displaying balanced levels of positive and negative work reflection (nonreflectors, low reflectors, moderate reflectors, and high reflectors). Analyses showed that persons differed significantly in energetic well-being depending on profile membership, with positive reflectors experiencing the highest well-being and negative reflectors experiencing the lowest well-being. Persons with balanced reflection profiles did not differ from one another in well-being. Our results provide new insights into the interplay of positive and negative work reflection during leisure time and its associations with employee well-being. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Test Performance Characteristics of the AIR, GAD-7, and HADS-Anxiety Screening Questionnaires for Anxiety in Chronic Obstructive Pulmonary Disease.

Rationale: Anxiety is a common comorbidity of chronic obstructive pulmonary disease (COPD) that is associated with higher morbidity and mortality. We evaluated three anxiety screening questionnaires: the Generalized Anxiety Disorder 7-Item Scale (GAD-7), the Hospital Anxiety and Depression Scale Anxiety subscale (HADS-A), and the Anxiety Inventory for Respiratory Disease (AIR).Objectives: To evaluate and compare the test performance characteristics of three anxiety screening questionnaires, using the Mini-International Neuropsychiatric Interview (MINI), version 7.0, as the "gold standard."Methods: Individuals with COPD were recruited at 16 centers. The MINI and questionnaires were administered by trained research coordinators at an in-person visit and readministered by telephone 2-4 weeks later. A composite score for the presence of any Diagnostic and Statistical Manual of Mental Disorders, 5th edition (DSM-V) anxiety disorder was computed, based on the MINI as the gold standard, compared with a participant screening positive on self-report measures for these analyses.Results: Two hundred and twenty eligible individuals with COPD were enrolled; 219 completed the study. Eleven percent were identified as having a DSM-V anxiety disorder, based on the MINI. Elevated anxiety symptoms based on questionnaires were 38% for the AIR, 30% for the GAD-7, and 20% for the HADS-A. Area under the receiver operating characteristic curve (AUC) was highest for the GAD-7 (0.78; 95% confidence interval [CI], 0.69-0.87), followed by the HADS-A (0.74; 95% CI, 0.64-0.84) and the AIR (0.66; 95% CI, 0.56-0.76). The AUC for the GAD-7 was significantly greater than for the AIR (P = 0.014). Sensitivity was not statistically different among the questionnaires: 77% for the GAD-7, 63% for the HADS-A, and 66% for the AIR. The HADS-A had the highest specificity, 85%, which was significantly higher than that of the GAD-7 (77%; P < 0.001) and the AIR (65%; P < 0.001); GAD-7 specificity was higher than AIR specificity (P < 0.001).Conclusions: Symptoms of anxiety among patients with COPD as identified by screening questionnaires were common and significantly higher than the prevalence of anxiety disorder meeting DSM-V criteria. The GAD-7, the HADS-A and the AIR questionnaires had fair to moderate psychometric properties as screening tools for anxiety in individuals with COPD, indicating the need for improved measures for this patient population.

BRCA1 is required for common-fragile-site stability via its G2/M checkpoint function.

Common fragile sites are loci that form chromosome gaps or breaks when DNA synthesis is partially inhibited. Fragile sites are prone to deletions, translocations, and other rearrangements that can cause the inactivation of associated tumor suppressor genes in cancer cells. It was previously shown that ATR is critical to fragile-site stability and that ATR-deficient cells have greatly elevated fragile-site expression (A. M. Casper, P. Nghiem, M. F. Arlt, and T. W. Glover, Cell 111:779-789, 2002). Here we demonstrate that mouse and human cells deficient for BRCA1, due to mutation or knockdown by RNA interference, also have elevated fragile-site expression. We further show that BRCA1 functions in the induction of the G(2)/M checkpoint after aphidicolin-induced replication stalling and that this checkpoint function is involved in fragile-site stability. These data indicate that BRCA1 is important in fragile-site stability and that fragile sites are recognized by the G(2)/M checkpoint pathway, in which BRCA1 plays a key role. Furthermore, they suggest that mutations in BRCA1 or interacting proteins could lead to rearrangements at fragile sites in cancer cells.

Advances in recovery research: What have we learned? What should be done next?

Job-stress recovery during nonwork time is an important factor for employee well-being. This article reviews the recovery literature, starting with a brief historical overview. It provides a definition of recovery that differentiates between recovery as a process and recovery as an outcome. Empirical studies have shown that recovery activities (e.g., physical exercise) and recovery experiences (e.g., psychological detachment from work) are negatively associated with strain symptoms (e.g., exhaustion) and positively associated with positive well-being indicators (e.g., vigor). Recovery activities and recovery experiences suffer when employees face a high level of job stressors. Psychological mechanisms underlying recovery seem to be similar across different temporal recovery settings (e.g., work breaks, free evenings, vacations) and seem to be enhanced in natural environments. Intervention studies have pointed to a diverse set of strategies for how everyday job-stress recovery can be supported. This article discusses 5 avenues for future research, with a particular focus on individual and contextual factors that may influence recovery as well as highlighting more complex temporal patterns than those uncovered in previous research. (PsycINFO Database Record

Remarkably Long-Tract Gene Conversion Induced by Fragile Site Instability in Saccharomyces cerevisiae.

Replication stress causes breaks at chromosomal locations called common fragile sites. Deletions causing loss of heterozygosity (LOH) in human tumors are strongly correlated with common fragile sites, but the role of gene conversion in LOH at fragile sites in tumors is less well studied. Here, we investigated gene conversion stimulated by instability at fragile site FS2 in the yeast Saccharomyces cerevisiae In our screening system, mitotic LOH events near FS2 are identified by production of red/white sectored colonies. We analyzed single nucleotide polymorphisms between homologs to determine the cause and extent of LOH. Instability at FS2 increases gene conversion 48- to 62-fold, and conversions unassociated with crossover represent 6-7% of LOH events. Gene conversion can result from repair of mismatches in heteroduplex DNA during synthesis-dependent strand annealing (SDSA), double-strand break repair (DSBR), and from break-induced replication (BIR) that switches templates [double BIR (dBIR)]. It has been proposed that SDSA and DSBR typically result in shorter gene-conversion tracts than dBIR. In cells under replication stress, we found that bidirectional tracts at FS2 have a median length of 40.8 kb and a wide distribution of lengths; most of these tracts are not crossover-associated. Tracts that begin at the fragile site FS2 and extend only distally are significantly shorter. The high abundance and long length of noncrossover, bidirectional gene-conversion tracts suggests that dBIR is a prominent mechanism for repair of lesions at FS2, thus this mechanism is likely to be a driver of common fragile site-stimulated LOH in human tumors.

Sites of genetic instability in mitosis and cancer.

Certain chromosomal regions called common fragile sites are prone to difficulty during replication. Many tumors have been shown to contain alterations at fragile sites. Several models have been proposed to explain why these sites are unstable. Here we describe work to investigate models of fragile site instability using a yeast artificial chromosome carrying human DNA from a common fragile site region. In addition, we describe a yeast system to investigate whether repair of breaks at a naturally occurring fragile site in yeast, FS2, involves mitotic recombination between homologous chromosomes, leading to loss of heterozygosity (LOH). Our initial evidence is that repair of yeast fragile site breaks does lead to LOH, suggesting that human fragile site breaks may similarly contribute to LOH in cancer. This work is focused on gaining understanding that may enable us to predict and prevent the situations and environments that promote genetic changes that contribute to tumor progression.

Chromosome aberrations resulting from double-strand DNA breaks at a naturally occurring yeast fragile site composed of inverted ty elements are independent of Mre11p and Sae2p.

Genetic instability at palindromes and spaced inverted repeats (IRs) leads to chromosome rearrangements. Perfect palindromes and IRs with short spacers can extrude as cruciforms or fold into hairpins on the lagging strand during replication. Cruciform resolution produces double-strand breaks (DSBs) with hairpin-capped ends, and Mre11p and Sae2p are required to cleave the hairpin tips to facilitate homologous recombination. Fragile site 2 (FS2) is a naturally occurring IR in Saccharomyces cerevisiae composed of a pair of Ty1 elements separated by approximately 280 bp. Our results suggest that FS2 forms a hairpin, rather than a cruciform, during replication in cells with low levels of DNA polymerase. Cleavage of this hairpin results in a recombinogenic DSB. We show that DSB formation at FS2 does not require Mre11p, Sae2p, Rad1p, Slx4p, Pso2p, Exo1p, Mus81p, Yen1p, or Rad27p. Also, repair of DSBs by homologous recombination is efficient in mre11 and sae2 mutants. Homologous recombination is impaired at FS2 in rad52 mutants and most aberrations reflect either joining of two broken chromosomes in a "half crossover" or telomere capping of the break. In support of hairpin formation precipitating DSBs at FS2, two telomere-capped deletions had a breakpoint near the center of the IR. In summary, Mre11p and Sae2p are not required for DSB formation at FS2 or the subsequent repair of these DSBs.