Interaction of yeast Rad51 and Rad52 relieves Rad52-mediated inhibition of de novo telomere addition.

DNA double-strand breaks (DSBs) are toxic forms of DNA damage that must be repaired to maintain genome integrity. Telomerase can act upon a DSB to create a de novo telomere, a process that interferes with normal repair and creates terminal deletions. We previously identified sequences in Saccharomyces cerevisiae (SiRTAs; Sites of Repair-associated Telomere Addition) that undergo unusually high frequencies of de novo telomere addition, even when the original chromosome break is several kilobases distal to the eventual site of telomerase action. Association of the single-stranded telomere binding protein Cdc13 with a SiRTA is required to stimulate de novo telomere addition. Because extensive resection must occur prior to Cdc13 binding, we utilized these sites to monitor the effect of proteins involved in homologous recombination. We find that telomere addition is significantly reduced in the absence of the Rad51 recombinase, while loss of Rad52, required for Rad51 nucleoprotein filament formation, has no effect. Deletion of RAD52 suppresses the defect of the rad51Δ strain, suggesting that Rad52 inhibits de novo telomere addition in the absence of Rad51. The ability of Rad51 to counteract this effect of Rad52 does not require DNA binding by Rad51, but does require interaction between the two proteins, while the inhibitory effect of Rad52 depends on its interaction with Replication Protein A (RPA). Intriguingly, the genetic interactions we report between RAD51 and RAD52 are similar to those previously observed in the context of checkpoint adaptation. Forced recruitment of Cdc13 fully restores telomere addition in the absence of Rad51, suggesting that Rad52, through its interaction with RPA-coated single-stranded DNA, inhibits the ability of Cdc13 to bind and stimulate telomere addition. Loss of the Rad51-Rad52 interaction also stimulates a subset of Rad52-dependent microhomology-mediated repair (MHMR) events, consistent with the known ability of Rad51 to prevent single-strand annealing.

"They Have Shown Me It Is Possible to Thrive within STEM": Incorporating Learning Assistants in General Chemistry Enhances Student Belonging and Confidence.

Students often experience social and psychological barriers to success in General Chemistry, which is a key gateway to many students' science pathways. Learning assistants (LAs) have the potential to reduce these barriers and to strengthen students' sense of belonging in General Chemistry and STEM more broadly. Here, we used a 17-item Likert scale to determine whether incorporating LAs into General Chemistry I and II enhances students' sense of belonging in these courses. The incorporation of LAs into General Chemistry I had a significant positive effect and a medium to large effect size for students in all student groups examined: women and men; students in both racially and ethnically underrepresented and well-represented groups; first- and continuing-generation students. In General Chemistry II, similar results were observed for women and men; students in well-represented racial and ethnic groups; continuing-generation students. Further, we asked students to reflect on the impact that working with LAs had on their sense of belonging in STEM and confidence in talking about science. Sixty percent of students indicated that working with LAs had a positive impact on their STEM belonging, with five themes describing LA impacts: reducing isolation, serving as inspirational role models, providing mentoring, increasing opportunities for engagement and confidence building, and serving as accessible and approachable sources of support. Sixty-one percent of students also indicated that working with LAs increased their confidence in talking about science, with three themes emerging: fostering an environment with a lower risk of negative judgment, providing increased opportunities for feedback, and supporting students as they practiced their growing skills. Together, these results indicate that LAs can be an important means to reduce social and psychological barriers for students in gateway science courses, increasing their sense that they belong to the class and STEM more broadly.

Yeast require redox switching in DNA primase.

Eukaryotic DNA primases contain a [4Fe4S] cluster in the C-terminal domain of the p58 subunit (p58C) that affects substrate affinity but is not required for catalysis. We show that, in yeast primase, the cluster serves as a DNA-mediated redox switch governing DNA binding, just as in human primase. Despite a different structural arrangement of tyrosines to facilitate electron transfer between the DNA substrate and [4Fe4S] cluster, in yeast, mutation of tyrosines Y395 and Y397 alters the same electron transfer chemistry and redox switch. Mutation of conserved tyrosine 395 diminishes the extent of p58C participation in normal redox-switching reactions, whereas mutation of conserved tyrosine 397 causes oxidative cluster degradation to the [3Fe4S]+ species during p58C redox signaling. Switching between oxidized and reduced states in the presence of the Y397 mutations thus puts primase [4Fe4S] cluster integrity and function at risk. Consistent with these observations, we find that yeast tolerate mutations to Y395 in p58C, but the single-residue mutation Y397L in p58C is lethal. Our data thus show that a constellation of tyrosines for protein-DNA electron transfer mediates the redox switch in eukaryotic primases and is required for primase function in vivo.

Emerging non-canonical roles for the Rad51-Rad52 interaction in response to double-strand breaks in yeast.

DNA double-strand break repair allows cells to survive both exogenous and endogenous insults to the genome. In yeast, the recombinases Rad51 and Rad52 are central to multiple forms of homology-dependent repair. Classically, Rad51 and Rad52 are thought to act cooperatively, with formation of the functional Rad51 nucleofilament facilitated by the mediator function of Rad52. Several studies have now identified functions for the interaction between Rad51 and Rad52 that are independent of the mediator function of Rad52 and affect a seemingly diverse array of functions in de novo telomere addition, global chromosome mobility following DNA damage, Rad51 nucleofilament stability, checkpoint adaptation, and microhomology-mediated chromosome rearrangements. Here, we review these functions with an emphasis on our recent discovery that the Rad51-Rad52 interaction influences the probability of de novo telomere addition at sites preferentially targeted by telomerase following a double-strand break (DSB). We present data addressing the prevalence of sites within the yeast genome that are capable of stimulating de novo telomere addition following a DSB and speculate about the potential role such sites may play in genome stability.

Pif1 family helicases suppress genome instability at G-quadruplex motifs.

The Saccharomyces cerevisiae Pif1 helicase is the prototypical member of the Pif1 DNA helicase family, which is conserved from bacteria to humans. Here we show that exceptionally potent G-quadruplex unwinding is conserved among Pif1 helicases. Moreover, Pif1 helicases from organisms separated by more than 3 billion years of evolution suppressed DNA damage at G-quadruplex motifs in yeast. The G-quadruplex-induced damage generated in the absence of Pif1 helicases led to new genetic and epigenetic changes. Furthermore, when expressed in yeast, human PIF1 suppressed both G-quadruplex-associated DNA damage and telomere lengthening.

Attitudes toward Tobacco, Alcohol, and Non-Alcoholic Beverage Advertisement Themes among Adolescent Boys.

BACKGROUND: Previous studies have examined what adolescents find appealing in tobacco and alcohol advertisements and how different themes in advertisements are used to manipulate consumer behaviors. Yet, we know little about the relationship between the themes portrayed in advertisements and youth attitudes towards those themes. OBJECTIVES: This study compared attitudes towards advertisements for different consumer products in a sample of urban and rural adolescent boys in order to examine how key marketing themes impact adolescent attitudes towards those advertisements. METHODS: Participants were 11- to 16-year-old boys (N = 1220) residing in either urban or rural Ohio Appalachian counties. Each participant viewed five print advertisements (one each for cigarettes, electronic cigarettes (e-cigarettes), smokeless tobacco (SLT), non-alcoholic beverages, and alcohol), presented in a random order, for eight seconds each. All advertisements had appeared in magazines that adolescent males commonly read. Attitudes towards each of the five advertisements were assessed. The advertisements were then coded for the presence of various themes, including social acceptance and masculinity. Analyses were conducted to determine associations between advertisement type and the attitude measure, and between the presence of a theme and the attitude measure. RESULTS: Overall, participants preferred non-tobacco advertisements to tobacco advertisements, rural participants had less positive attitudes and participants who had peers who used tobacco had more positive attitudes. Social acceptance and entertainment themes increased the appeal of SLT advertisements, and sex appeal increased the appeal of e-cigarette advertisements. Conclusions/Importance: Findings suggest that advertisements that promote the social nature of use in SLT advertisements may be of particular concern for their influence on adolescent boys.

SV40 utilizes ATM kinase activity to prevent non-homologous end joining of broken viral DNA replication products.

Simian virus 40 (SV40) and cellular DNA replication rely on host ATM and ATR DNA damage signaling kinases to facilitate DNA repair and elicit cell cycle arrest following DNA damage. During SV40 DNA replication, ATM kinase activity prevents concatemerization of the viral genome whereas ATR activity prevents accumulation of aberrant genomes resulting from breakage of a moving replication fork as it converges with a stalled fork. However, the repair pathways that ATM and ATR orchestrate to prevent these aberrant SV40 DNA replication products are unclear. Using two-dimensional gel electrophoresis and Southern blotting, we show that ATR kinase activity, but not DNA-PK(cs) kinase activity, facilitates some aspects of double strand break (DSB) repair when ATM is inhibited during SV40 infection. To clarify which repair factors associate with viral DNA replication centers, we examined the localization of DSB repair proteins in response to SV40 infection. Under normal conditions, viral replication centers exclusively associate with homology-directed repair (HDR) and do not colocalize with non-homologous end joining (NHEJ) factors. Following ATM inhibition, but not ATR inhibition, activated DNA-PK(cs) and KU70/80 accumulate at the viral replication centers while CtIP and BLM, proteins that initiate 5' to 3' end resection during HDR, become undetectable. Similar to what has been observed during cellular DSB repair in S phase, these data suggest that ATM kinase influences DSB repair pathway choice by preventing the recruitment of NHEJ factors to replicating viral DNA. These data may explain how ATM prevents concatemerization of the viral genome and promotes viral propagation. We suggest that inhibitors of DNA damage signaling and DNA repair could be used during infection to disrupt productive viral DNA replication.

Normal telomere length maintenance in Saccharomyces cerevisiae requires nuclear import of the ever shorter telomeres 1 (Est1) protein via the importin alpha pathway.

The Est1 (ever shorter telomeres 1) protein is an essential component of yeast telomerase, a ribonucleoprotein complex that restores the repetitive sequences at chromosome ends (telomeres) that would otherwise be lost during DNA replication. Previous work has shown that the telomerase RNA component (TLC1) transits through the cytoplasm during telomerase biogenesis, but mechanisms of protein import have not been addressed. Here we identify three nuclear localization sequences (NLSs) in Est1p. Mutation of the most N-terminal NLS in the context of full-length Est1p reduces Est1p nuclear localization and causes telomere shortening-phenotypes that are rescued by fusion with the NLS from the simian virus 40 (SV40) large-T antigen. In contrast to that of the TLC1 RNA, Est1p nuclear import is facilitated by Srp1p, the yeast homolog of importin α. The reduction in telomere length observed at the semipermissive temperature in a srp1 mutant strain is rescued by increased Est1p expression, consistent with a defect in Est1p nuclear import. These studies suggest that at least two nuclear import pathways are required to achieve normal telomere length homeostasis in yeast.

A mutation in the catalytic subunit of yeast telomerase alters primer-template alignment while promoting processivity and protein-DNA binding.

Telomerase is a ribonucleoprotein complex that is required for maintenance of linear chromosome ends (telomeres). In yeast, the Est2 protein reverse transcribes a short template region of the TLC1 RNA using the chromosome terminus to prime replication. Yeast telomeres contain heterogeneous G(1-3)T sequences that arise from incomplete reverse transcription of the TLC1 template and alignment of the DNA primer at multiple sites within the template region. We have previously described mutations in the essential N-terminal TEN domain of Est2p that alter telomere sequences. Here, we demonstrate that one of these mutants, glutamic acid 76 to lysine (est2-LT(E76K)), restricts possible alignments between the DNA primer and the TLC1 template. In addition, this mutant exhibits increased processivity in vivo. Within the context of the telomerase enzyme, the Est2p TEN domain is thought to contribute to enzyme processivity by mediating an anchor-site interaction with the DNA primer. We show that binding of the purified TEN domain (residues 1-161) to telomeric DNA is enhanced by the E76K mutation. These results support the idea that the anchor-site interaction contributes to telomerase processivity and suggest a role for the anchor site of yeast telomerase in mediating primer-template alignment within the active site.

Hotspot of de novo telomere addition stabilizes linear amplicons in yeast grown in sulfate-limiting conditions.

Evolution is driven by the accumulation of competing mutations that influence survival. A broad form of genetic variation is the amplification or deletion of DNA (≥50 bp) referred to as copy number variation (CNV). In humans, CNV may be inconsequential, contribute to minor phenotypic differences, or cause conditions such as birth defects, neurodevelopmental disorders, and cancers. To identify mechanisms that drive CNV, we monitored the experimental evolution of Saccharomyces cerevisiae populations grown under sulfate-limiting conditions. Cells with increased copy number of the gene SUL1, which encodes a primary sulfate transporter, exhibit a fitness advantage. Previously, we reported interstitial inverted triplications of SUL1 as the dominant rearrangement in a haploid population. Here, in a diploid population, we find instead that small linear fragments containing SUL1 form and are sustained over several generations. Many of the linear fragments are stabilized by de novo telomere addition within a telomere-like sequence near SUL1 (within the SNF5 gene). Using an assay that monitors telomerase action following an induced chromosome break, we show that this region acts as a hotspot of de novo telomere addition and that required sequences map to a region of <250 base pairs. Consistent with previous work showing that association of the telomere-binding protein Cdc13 with internal sequences stimulates telomerase recruitment, mutation of a four-nucleotide motif predicted to associate with Cdc13 abolishes de novo telomere addition. Our study suggests that internal telomere-like sequences that stimulate de novo telomere addition can contribute to adaptation by promoting genomic plasticity.

A comprehensive map of hotspots of de novo telomere addition in Saccharomyces cerevisiae.

Telomere healing occurs when telomerase, normally restricted to chromosome ends, acts upon a double-strand break to create a new, functional telomere. De novo telomere addition on the centromere-proximal side of a break truncates the chromosome but, by blocking resection, may allow the cell to survive an otherwise lethal event. We previously identified several sequences in the baker’s yeast, Saccharomyces cerevisiae , that act as hotspots of de novo telomere addition (termed Sites of Repair-associated Telomere Addition or SiRTAs), but the distribution and functional relevance of SiRTAs is unclear. Here, we describe a high-throughput sequencing method to measure the frequency and location of telomere addition within sequences of interest. Combining this methodology with a computational algorithm that identifies SiRTA sequence motifs, we generate the first comprehensive map of telomere-addition hotspots in yeast. Putative SiRTAs are strongly enriched in subtelomeric regions where they may facilitate formation of a new telomere following catastrophic telomere loss. In contrast, outside of subtelomeres, the distribution and orientation of SiRTAs appears random. Since truncating the chromosome at most SiRTAs would be lethal, this observation argues against selection for these sequences as sites of telomere addition per se. We find, however, that sequences predicted to function as SiRTAs are significantly more prevalent across the genome than expected by chance. Sequences identified by the algorithm bind the telomeric protein Cdc13, raising the possibility that association of Cdc13 with single-stranded regions generated during the response to DNA damage may facilitate DNA repair more generally.

A comprehensive map of hotspots of de novo telomere addition in Saccharomyces cerevisiae.

Telomere healing occurs when telomerase, normally restricted to chromosome ends, acts upon a double-strand break to create a new, functional telomere. De novo telomere addition (dnTA) on the centromere-proximal side of a break truncates the chromosome but, by blocking resection, may allow the cell to survive an otherwise lethal event. We previously identified several sequences in the baker's yeast, Saccharomyces cerevisiae, that act as hotspots of dnTA [termed Sites of Repair-associated Telomere Addition (SiRTAs)], but the distribution and functional relevance of SiRTAs is unclear. Here, we describe a high-throughput sequencing method to measure the frequency and location of telomere addition within sequences of interest. Combining this methodology with a computational algorithm that identifies SiRTA sequence motifs, we generate the first comprehensive map of telomere-addition hotspots in yeast. Putative SiRTAs are strongly enriched in subtelomeric regions where they may facilitate formation of a new telomere following catastrophic telomere loss. In contrast, outside of subtelomeres, the distribution and orientation of SiRTAs appears random. Since truncating the chromosome at most SiRTAs would be lethal, this observation argues against selection for these sequences as sites of telomere addition per se. We find, however, that sequences predicted to function as SiRTAs are significantly more prevalent across the genome than expected by chance. Sequences identified by the algorithm bind the telomeric protein Cdc13, raising the possibility that association of Cdc13 with single-stranded regions generated during the response to DNA damage may facilitate DNA repair more generally.

Stimulation of yeast telomerase activity by the ever shorter telomere 3 (Est3) subunit is dependent on direct interaction with the catalytic protein Est2.

Telomerase is a multisubunit enzyme that maintains genome stability through its role in telomere replication. Although the Est3 protein is long recognized as an essential telomerase component, how it associates with and functions in the telomerase complex has remained enigmatic. Here we provide the first evidence of a direct interaction between Saccharomyces cerevisiae Est3p and the catalytic protein subunit (Est2p) by demonstrating that recombinant Est3p binds the purified telomerase essential N-terminal (TEN) domain of Est2p in vitro. Mutations in a small cluster of amino acids predicted to lie on the surface of Est3p disrupt this interaction with Est2p, reduce assembly of Est3p with telomerase in vivo, and cause telomere shortening and senescence. We also show that recombinant Est3p stimulates telomerase activity above basal levels in vitro in a manner dependent on the Est2p TEN domain interaction. Together, these results define a direct binding interaction between Est3p and Est2p and reconcile the effect of S. cerevisiae Est3p with previous experiments showing that Est3p homologs in related yeast species influence telomerase activity. Additionally, it contributes functional support to the idea that Est3p is structurally related to the mammalian shelterin protein, TPP1, which also influences telomerase activity through interaction with the Est2p homolog, TERT.

"It made me feel like a bigger part of the STEM community": Incorporation of Learning Assistants Enhances Students' Sense of Belonging in a Large Introductory Biology Course.

Large introductory science courses are a particularly important and challenging target for creating inclusive learning environments. In this study, we examined the impact of incorporating learning assistants (LAs) on the learning environment in an introductory biology course taught with two different structures: an in-person lecture with intermittent active-learning components and an online setting taught with a flipped instructional approach. Using a survey that measured sense of belonging in a single class, we found that students in sections with LAs reported greater sense of belonging than students in sections without LAs in both class structures. Further, student focus groups revealed that LAs promoted learning and engagement in the class by answering questions and providing clarity; allowing more use of active- and interactive-learning structures; and serving as accessible, approachable, and immediate sources of help. Student responses also indicated that LAs promoted a sense of belonging in science, technology, engineering, and mathematics (STEM) by decreasing feelings of isolation, serving as inspirational role models, clarifying progression through the STEM educational system, and helping students become more engaged and confident in their STEM-related knowledge and skills. These findings indicate that LAs can support multiple elements of inclusive STEM learning environments.

Proteasome-dependent degradation of Est1p regulates the cell cycle-restricted assembly of telomerase in Saccharomyces cerevisiae.

Telomerase counteracts loss of terminal sequences incurred during DNA replication. In S. cerevisiae, telomerase contains an RNA template (TLC1), a reverse transcriptase (Est2p) and at least two regulatory proteins (Est1p and Est3p). Whereas Est2p is constitutively telomere bound, Est1p associates in late S phase, coincident with telomere lengthening. Here we directly demonstrate by coimmunoprecipitation that the composition of telomerase varies during the cell cycle. The absence of Est1p and Est3p from the complex during G1 phase can be attributed to proteasome-dependent degradation of Est1p. Stabilization of Est1p during G1 phase promotes telomerase assembly, revealing a previously uncharacterized role for Est1p in the recruitment of Est3p to the telomerase complex. Though catalytically active, complexes assembled during G1 cannot lengthen telomeres. We conclude that telomerase assembly during G1 phase is regulated by Est1p stability, but assembly is insufficient to activate telomerase at telomeres.

When the Ends Justify the Means: Regulation of Telomere Addition at Double-Strand Breaks in Yeast.

Telomeres, repetitive sequences located at the ends of most eukaryotic chromosomes, provide a mechanism to replenish terminal sequences lost during DNA replication, limit nucleolytic resection, and protect chromosome ends from engaging in double-strand break (DSB) repair. The ribonucleoprotein telomerase contains an RNA subunit that serves as the template for the synthesis of telomeric DNA. While telomere elongation is typically primed by a 3' overhang at existing chromosome ends, telomerase can act upon internal non-telomeric sequences. Such de novo telomere addition can be programmed (for example, during chromosome fragmentation in ciliated protozoa) or can occur spontaneously in response to a chromosome break. Telomerase action at a DSB can interfere with conservative mechanisms of DNA repair and results in loss of distal sequences but may prevent additional nucleolytic resection and/or chromosome rearrangement through formation of a functional telomere (termed "chromosome healing"). Here, we review studies of spontaneous and induced DSBs in the yeast Saccharomyces cerevisiae that shed light on mechanisms that negatively regulate de novo telomere addition, in particular how the cell prevents telomerase action at DSBs while facilitating elongation of critically short telomeres. Much of our understanding comes from the use of perfect artificial telomeric tracts to "seed" de novo telomere addition. However, endogenous sequences that are enriched in thymine and guanine nucleotides on one strand (TG-rich) but do not perfectly match the telomere consensus sequence can also stimulate unusually high frequencies of telomere formation following a DSB. These observations suggest that some internal sites may fully or partially escape mechanisms that normally negatively regulate de novo telomere addition.

Parental Status and Biological Functioning: Findings from the Nashville Stress and Health Study.

Does childrearing affect the biological functioning of parents? To address this question, we analyze cross-sectional survey and biomarker data from Vanderbilt University's Nashville Stress and Health Study, a probability sample of non-Hispanic white and black working-age adults from Davidson County, Tennessee (2011-2014; n = 1,252). Multivariable regression analyses reveal a linear dose-response relationship between the number of children living in a respondent's home and (a) increased allostatic load, and (b) decreased leukocyte telomere length. We found no differences in biological functioning between childless respondents and empty-nest parents. These findings also withstood controls for a battery of socioeconomic factors. The implications of these findings and suggestions for future research are discussed.

Regulation of telomere length by an N-terminal region of the yeast telomerase reverse transcriptase.

Telomerase is a reverse transcriptase that maintains chromosome integrity through synthesis of repetitive telomeric sequences on the ends of eukaryotic chromosomes. In the yeast Saccharomyces cerevisiae, telomere length homeostasis is achieved through negative regulation of telomerase access to the chromosome terminus by telomere-bound Rap1 protein and its binding partners, Rif1p and Rif2p, and positive regulation by factors such as Ku70/80, Tel1p, and Cdc13p. Here we report the identification of mutations within an N-terminal region (region I) of the yeast telomerase catalytic subunit (Est2p) that cause telomere lengthening without altering measurable catalytic properties of the enzyme in vitro. These telomerase mutations affect telomere length through a Ku-independent mechanism and do not alter chromosome end structure. While Tel1p is required for expression of the telomere-lengthening phenotype, Rif1p and Rif2p are not, suggesting that telomere overextension is independent of Rap1p. Taken together, these data suggest that specific amino acids within region I of the catalytic subunit of yeast telomerase play a previously unanticipated role in the response to Tel1p regulation at the telomere.

Maize centromere mapping: a comparison of physical and genetic strategies.

Centromere positions on 7 maize chromosomes were compared on the basis of data from 4 to 6 mapping techniques per chromosome. Centromere positions were first located relative to molecular markers by means of radiation hybrid lines and centric fission lines recovered from oat-maize chromosome addition lines. These centromere positions were then compared with new data from centric fission lines recovered from maize plants, half-tetrad mapping, and fluorescence in situ hybridizations and to data from earlier studies. Surprisingly, the choice of mapping technique was not the critical determining factor. Instead, on 4 chromosomes, results from all techniques were consistent with a single centromere position. On chromosomes 1, 3, and 6, centromere positions were not consistent even in studies using the same technique. The conflicting centromere map positions on chromosomes 1, 3, and 6 could be explained by pericentric inversions or alternative centromere positions on these chromosomes.

Perceived Unfair Treatment by Police, Race, and Telomere Length: A Nashville Community-based Sample of Black and White Men.

Police maltreatment, whether experienced personally or indirectly through one's family or friends, represents a structurally rooted public health problem that disproportionately affects minorities. Researchers, however, know little about the physiological mechanisms connecting unfair treatment by police (UTBP) to poor health. Shortened telomeres due to exposure to this stressor represent one plausible mechanism. Using data from a community sample of black (n = 262) and white (n = 252) men residing in Nashville-Davidson County, we test four hypotheses: (1) Black men will be more likely to report UTBP than white men, (2) those reporting UTBP will have shorter telomeres than those not reporting UTBP, (3) this association will be more pronounced among black men, and (4) these hypotheses will extend to those who report vicarious UTBP. Results reveal support for all hypotheses. The implications for our findings are discussed as they pertain to debates on policing practices and health disparities research.