84 research outputs found
The Impact of Empathy Skills Training on Middle School Children
This Master’s thesis explored the subject of empathy in adolescents. The purpose of this research project was to examine if empathy skills training has a positive effect on empathy skill development in middle school children. This study was an outcome-based program evaluation with a pre/post test design. In this quantitative study, a pre/post test scale was developed by the researcher to assess students’ experiences with empathy within the school context before and after an eight session intervention including activities. Fourteen eighth grade students from a middle school in Western New York participated after consent was requested from 30 parents or guardians. The researcher shared a descriptive analysis of the study and results based on data from surveys administered by the researcher prior to the program intervention and then a post-survey following the eight sessions together. The results of the study indicated a heightened awareness of bullying as well as implications of actions by self and others. Follow-up programs are suggested for future research
PIF1 HELICASE AND POLYMERASE ZETA (ζ) CHARACTERIZE TWO PATHWAYS OF MUTAGENESIS ASSOCIATED WITH BREAK INDUCED REPLICATION.
poster abstractThe fidelity of DNA synthesis differs among the various processes in which it is involved. While normal S-phase DNA replication is highly accu-rate, DNA synthesis associated with DNA repair is often error-prone. Recent-ly, we have analyzed the accuracy of Break-induced replication, which is a unique cellular process that mimics normal DNA replication in its processivity and rate, but is initiated at double-strand breaks (DSBs) rather than at repli-cation origins. We have demonstrated that BIR is associated with approxi-mately a thousand-fold increase of the rate of frameshift mutations as com-pared to spontaneous events. Here we have identified 5’ – 3’ helicase Pif1p and translesion polymerase Pol ζ as the two major components in promoting frameshift mutations associated with BIR. We have also employed a rever-sion assay using base substitution reporter ura 3-29 to demonstrate that BIR elevates base substitution mutations by a fold of 400 over normal DNA repli-cation. This mutagenic character led us to explore the mode of repair synthesis associated with BIR.Our data suggests that BIR maybe following an unusual, conservative mode of synthesis very different from the usual semiconserva-tive mode of synthesis followed by normal S-phase DNA replication
Sulfolobus Mutants, Generated via PCR Products, Which Lack Putative Enzymes of UV Photoproduct Repair
In order to determine the biological relevance of two S. acidocaldarius proteins to the repair of UV photoproducts, the corresponding genes (Saci_1227 and Saci_1096) were disrupted, and the phenotypes of the resulting mutants were examined by various genetic assays. The disruption used integration by homologous recombination of a functional but heterologous pyrE gene, promoted by short sequences attached to both ends via PCR. The phenotypic analyses of the disruptants confirmed that ORF Saci_1227 encodes a DNA photolyase which functions in vivo, but they could not implicate ORF Saci_1096 in repair of UV- or other externally induced DNA damage despite its similarity to genes encoding UV damage endonucleases. The success of the gene-disruption strategy, which used 5′ extensions of PCR primers to target cassette integration, suggests potential advantages for routine construction of Sulfolobus strains
MMBIRFinder: A Tool to Detect Microhomology-Mediated Break-Induced Replication
The introduction of next-generation sequencing technologies has radically changed the way we view structural genetic events. Microhomology-mediated break-induced replication (MMBIR) is just one of the many mechanisms that can cause genomic destabilization that may lead to cancer. Although the mechanism for MMBIR remains unclear, it has been shown that MMBIR is typically associated with template-switching events. Currently, to our knowledge, there is no existing bioinformatics tool to detect these template-switching events. We have developed MMBIRFinder, a method that detects template-switching events associated with MMBIR from whole-genome sequenced data. MMBIRFinder uses a half-read alignment approach to identify potential regions of interest. Clustering of these potential regions helps narrow the search space to regions with strong evidence. Subsequent local alignments identify the template-switching events with single-nucleotide accuracy. Using simulated data, MMBIRFinder identified 83 percent of the MMBIR regions within a five nucleotide tolerance. Using real data, MMBIRFinder identified 16 MMBIR regions on a normal breast tissue data sample and 51 MMBIR regions on a triple-negative breast cancer tumor sample resulting in detection of 37 novel template-switching events. Finally, we identified template-switching events residing in the promoter region of seven genes that have been implicated in breast cancer
APOBEC3 genes: retroviral restriction factors to cancer drivers
The APOBEC3 cytosine deaminases play key roles in innate immunity through their ability to mutagenize viral DNA and restrict viral replication. Recent advances in cancer genomics, together with biochemical characterization of the APOBEC3 enzymes, have now implicated at least two family members in somatic mutagenesis during tumor development. We review the evidence linking these enzymes to carcinogenesis and highlight key questions, including the potential mechanisms that misdirect APOBEC3 activity to the host genome, the links to viral infection, and the association between a common APOBEC3 polymorphism and cancer risk
Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways
Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies
Role of the Pif1-PCNA Complex in Pol δ-Dependent Strand Displacement DNA Synthesis and Break-Induced Replication
Break-induced replication is a source of mutation clusters underlying kataegis
Clusters of simultaneous multiple mutations can be a source of rapid change during carcinogenesis and evolution. Such mutation clusters have been recently shown to originate from DNA damage within long single-stranded DNA (ssDNA) formed at resected double-strand breaks and dysfunctional replication forks. Here, we identify double-strand break (DSB)-induced replication (BIR) as another powerful source of mutation clusters that formed in nearly half of wild-type yeast cells undergoing BIR in the presence of alkylating damage. Clustered mutations were primarily formed along the track of DNA synthesis and were frequently associated with additional breakage and rearrangements. Moreover, the base specificity, strand coordination, and strand bias of the mutation spectrum were consistent with mutations arising from damage in persistent ssDNA stretches within unconventional replication intermediates. Altogether, these features closely resemble kataegic events in cancers, suggesting that replication intermediates during BIR may be the most prominent source of mutation clusters across species
Repair of multiple simultaneous double-strand breaks causes bursts of genome-wide clustered hypermutation.
A single cancer genome can harbor thousands of clustered mutations. Mutation signature analyses have revealed that the origin of clusters are lesions in long tracts of single-stranded (ss) DNA damaged by apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases, raising questions about molecular mechanisms that generate long ssDNA vulnerable to hypermutation. Here, we show that ssDNA intermediates formed during the repair of gamma-induced bursts of double-strand breaks (DSBs) in the presence of APOBEC3A in yeast lead to multiple APOBEC-induced clusters similar to cancer. We identified three independent pathways enabling cluster formation associated with repairing bursts of DSBs: 5' to 3' bidirectional resection, unidirectional resection, and break-induced replication (BIR). Analysis of millions of mutations in APOBEC-hypermutated cancer genomes revealed that cancer tolerance to formation of hypermutable ssDNA is similar to yeast and that the predominant pattern of clustered mutagenesis is the same as in resection-defective yeast, suggesting that cluster formation in cancers is driven by a BIR-like mechanism. The phenomenon of genome-wide burst of clustered mutagenesis revealed by our study can play an important role in generating somatic hypermutation in cancers as well as in noncancerous cells
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