Browsing by Subject "DNA"

Now showing 1 - 10 of 48
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    Acetylation regulates DNA repair mechanisms in human cells
    (Informa UK (Taylor & Francis), 2016-06-02) Piekna-Przybylska, Dorota; Bambara, Robert A.; Balakrishnan, Lata; Department of Biology, School of Science
    The p300-mediated acetylation of enzymes involved in DNA repair and replication has been previously shown to stimulate or inhibit their activities in reconstituted systems. To explore the role of acetylation on DNA repair in cells we constructed plasmid substrates carrying inactivating damages in the EGFP reporter gene, which should be repaired in cells through DNA mismatch repair (MMR) or base excision repair (BER) mechanisms. We analyzed efficiency of repair within these plasmid substrates in cells exposed to deacetylase and acetyltransferase inhibitors, and also in cells deficient in p300 acetyltransferase. Our results indicate that protein acetylation improves DNA mismatch repair in MMR-proficient HeLa cells and also in MMR-deficient HCT116 cells. Moreover, results suggest that stimulated repair of mismatches in MMR-deficient HCT116 cells is done though a strand-displacement synthesis mechanism described previously for Okazaki fragments maturation and also for the EXOI-independent pathway of MMR. Loss of p300 reduced repair of mismatches in MMR-deficient cells, but did not have evident effects on BER mechanisms, including the long patch BER pathway. Hypoacetylation of the cells in the presence of acetyltransferase inhibitor, garcinol generally reduced efficiency of BER of 8-oxoG damage, indicating that some steps in the pathway are stimulated by acetylation.
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    Biogenesis and molecular characteristics of serum hepatitis B virus RNA
    (Public Library of Science, 2020-10-20) Shen, Sheng; Xie, Zhanglian; Cai, Dawei; Yu, Xiaoyang; Zhang, Hu; Kim, Elena S.; Zhou, Bin; Hou, Jinlin; Zhang, Xiaoyong; Huang, Qi; Sun, Jian; Guo, Haitao; Medicine, School of Medicine
    HBV is an enveloped DNA virus that replicates its DNA genome via reverse transcription of a pregenomic (pg) RNA intermediate in hepatocytes. Interestingly, HBV RNA can be detected in virus-like particles in chronic hepatitis B (CHB) patient serum and has been utilized as a biomarker for intrahepatic cccDNA activity in treated patients. However, the biogenesis and molecular characteristics of serum HBV RNA remain to be fully defined. In this study, we found that the encapsidated serum HBV RNA predominately consists of pgRNA, which are detergent- and ribonuclease-resistant. Through blocking HBV DNA replication without affecting pgRNA encapsidation by using the priming-defective HBV mutant Y63D or 3TC treatment, we demonstrated that the cell culture supernatant contains a large amount of pgRNA-containing nonenveloped capsids and a minor population of pgRNA-containing virions. The formation of pgRNA-virion requires both capsid assembly and viral envelope proteins, which can be inhibited by capsid assembly modulators and an envelope–knockout mutant, respectively. Furthermore, the pgRNA-virion utilizes the multivesicular body pathway for egress, in a similar way as DNA-virion morphogenesis. Northern blotting, RT-PCR, and 3’ RACE assays revealed that serum/supernatant HBV pgRNA are mainly spliced and devoid of the 3’-terminal sequences. Furthermore, pgRNA-virion collected from cells treated with a reversible HBV priming inhibitor L-FMAU was unable to establish infection in HepG2-NTCP cells. In summary, serum HBV RNA is secreted in noninfectious virion-like particle as spliced and poly(A)-free pgRNA. Our study will shed light on the molecular biology of serum HBV RNA in HBV life cycle, and aid the development of serum HBV RNA as a novel biomarker for CHB diagnosis and treatment prognosis.
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    Blood on FTA™ Paper: Does Punch Location Affect the Quality of a Forensic DNA Profile?
    (2013-03-06) Carter, Megan Elizabeth; Picard, Christine; Siegel, Jay A.; Randall, Stephen Karl, 1953-
    Forensic DNA profiling is widely used as an identification tool for associating an individual with evidence of a crime. Analysis of a DNA sample involves observation of data in the form of an electropherogram, and subsequently annotating a DNA “profile” from an individual or from the evidence. The profile obtained from the evidence can be compared to reference profiles deposited in a national DNA database, which may include the potential contributor. Following a match, a random match probability is calculated to determine how common that genotype is in the population. This is the probability of obtaining that same DNA profile by sampling from a pool of unrelated individuals. Each state has adopted various laws requiring suspects and/or offenders to submit a DNA sample for the national database (such as California’s law that all who are arrested must provide a DNA sample). These profiles can then be associated with past unsolved crimes, and remain in the database to be searched in the event of future crimes. In the case of database samples, a physical sample of the offender’s DNA must be kept on file in the laboratory indefinitely so that in the event of a database hit, the sample is able to be retested. Current methods are to collect a buccal swab or blood sample, and store the DNA extracts under strict preservation conditions, i.e. cold storage, typically -20° C. With continually increasing number of samples submitted, a burden is placed on crime labs to store these DNA extracts. A solution was required to help control the costs of properly storing the samples. FTA™ paper was created to fulfill the need for inexpensive, low maintenance, long term storage of biological samples, which makes it ideal for use with convicted offender DNA samples. FTA™ paper is a commercially produced, chemically treated paper that allows DNA to be stored at room temperature for years with no costly storage facilities or conditions. Once a sample is required for DNA testing, a small disc is removed and is to be used directly in a PCR reaction. A high quality profile is important for comparing suspect profiles to unknown or database profiles. A single difference between a suspect and evidentiary sample can lead to exclusion. Unfortunately, the DNA profile results yielded from the direct addition have been unfavorable. Thus, most crime laboratories will extract the DNA from the disc, leading to additional time and cost to analyze a reference sample. Many of the profiles from the direct addition of an FTA™ disc result in poor quality profiles, likely due to an increase in PCR inhibitors and high concentrations of DNA. Currently, standardized protocols regarding the recommended locations for removal of a sample disc from a bloodspot on an FTA™ card does not exist. This study aims to validate the optimal location by comparing DNA profiles obtained from discs removed from the center, halfway, and edge locations of a bloodspot from 50 anonymous donors. Optimal punch location was first scored on the number of failed, partial or discordant profiles. Then, profile quality was determined based on peak characteristics of the resulting DNA profiles. The results for all three disc locations were 5.3% failed amplifications, 4.2% partial amplifications, and one case of a discordant profile. Profile quality for the majority of the samples showed a high incidence of stutter and the absence of non-template adenylation. Of the three disc locations, the edge of the blood stain was ideal, due to a presumably lower concentration of DNA and likely more dilute amount of the PCR inhibitor heme. Therefore, based on the results of this study, there is a greater probability of success using a sample from the edge of a blood stain spotted in FTA™ paper than any other location of the FTA™ card.
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    Calcium-Sensing Receptor Genotype and Response to Cinacalcet in Patients Undergoing Hemodialysis
    (American Society of Nephrology, 2017-07-07) Moe, Sharon M.; Wetherill, Leah; Decker, Brian Scott; Lai, Dongbing; Abdalla, Safa; Long, Jin; Vatta, Matteo; Foroud, Tatiana M.; Chertow, Glenn M.; Medicine, School of Medicine
    BACKGROUND AND OBJECTIVES: We tested the hypothesis that single nucleotide polymorphisms (SNPs) in the calcium-sensing receptor (CASR) alter the response to the calcimimetic cinacalcet. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We analyzed DNA samples in the Evaluation of Cinacalcet HCl Therapy to Lower Cardiovascular Events (EVOLVE) trial, a randomized trial comparing cinacalcet to placebo on a background of usual care. Of the 3883 patients randomized, 1919 (49%) consented to DNA collection, and samples from 1852 participants were genotyped for 18 CASR polymorphisms. The European ancestry (EA; n=1067) and African ancestry (AfAn; n=405) groups were assessed separately. SNPs in CASR were tested for their association with biochemical measures of mineral metabolism at baseline, percent change from baseline to 20 weeks, and risk of clinical fracture as dependent variables. RESULTS: There were modest associations of CASR SNPs with increased baseline serum parathyroid hormone and bone alkaline phosphatase primarily with the minor allele in the EA group (all P≤0.03), but not in the AfAn sample. In contrast, there was a modest association of decreased baseline serum calcium and FGF23 with CASR SNPs (P=0.04) primarily with the minor allele in the AfAn but not in the EA sample. The minor allele of two SNPs was associated with decreased percent reduction in parathyroid hormone from baseline to 20 weeks in the EA population (P<0.04) and this was not altered with cinacalcet. In both EA and AfAn, the same SNP (rs9740) was associated with decreased calcium with cinacalcet treatment (EA and AfAn P≤0.03). Three SNPs in high linkage disequilibrium were associated with a higher risk of clinical fracture that was attenuated by cinacalcet treatment in the EA sample (P<0.04). CONCLUSIONS: These modest associations, if validated, may provide explanations for differences in CKD-mineral bone disorder observed in EA and AfAn populations, and for differential biochemical responses to calcimimetics.
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    Characterization of Reference Materials for CYP3A4 and CYP3A5: A GeT-RM Collaborative Project
    (Elsevier, 2023) Gaedigk, Andrea; Boone, Erin C.; Turner, Amy J.; van Schaik, Ron H.N.; Cheranova, Dilyara; Wang, Wendy Y.; Broeckel, Ulrich; Granfield, Caitlin A.; Hodge, Jennelle C.; Ly, Reynold C.; Lynnes, Ty C.; Mitchell, Matthew W.; Moyer, Ann M.; Oliva, Jason; Kalman, Lisa V.; Medical and Molecular Genetics, School of Medicine
    Pharmacogenetic testing for CYP3A4 is increasingly provided by clinical and research laboratories; however, only a limited number of quality control and reference materials are currently available for many of the CYP3A4 variants included in clinical tests. To address this need, the Division of Laboratory Systems, CDC-based Genetic Testing Reference Material Coordination Program (GeT-RM), in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Institute for Medical Research, has characterized 30 DNA samples derived from Coriell cell lines for CYP3A4. Samples were distributed to five volunteer laboratories for genotyping using a variety of commercially available and laboratory-developed tests. Sanger and next-generation sequencing were also utilized by some of the laboratories. Whole-genome sequencing data from the 1000 Genomes Projects were utilized to inform genotype. Twenty CYP3A4 alleles were identified in the 30 samples characterized for CYP3A4: CYP3A4∗4, ∗5, ∗6, ∗7, ∗8, ∗9, ∗10, ∗11, ∗12, ∗15, ∗16, ∗18, ∗19, ∗20, ∗21, ∗22, ∗23, ∗24, ∗35, and a novel allele, CYP3A4∗38. Nineteen additional samples with preexisting data for CYP3A4 or CYP3A5 were re-analyzed to generate comprehensive reference material panels for these genes. These publicly available and well-characterized materials can be used to support the quality assurance and quality control programs of clinical laboratories performing clinical pharmacogenetic testing.
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    Consecutive non-natural PZ nucleobase pairs in DNA impact helical structure as seen in 50 μs molecular dynamics simulations
    (Oxford University Press, 2017-04-20) Molt, Robert W.; Georgiadis, Millie M.; Richards, Nigel G. J.; Department of Biochemistry and Molecular Biology, School of Medicine
    Z: Little is known about the influence of multiple consecutive 'non-standard' ( , 6-amino-5-nitro-2(1H)-pyridone, and , 2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one) nucleobase pairs on the structural parameters of duplex DNA. nucleobase pairs follow standard rules for Watson-Crick base pairing but have rearranged hydrogen bonding donor and acceptor groups. Using the X-ray crystal structure as a starting point, we have modeled the motions of a DNA duplex built from a self-complementary oligonucleotide (5΄-CTTATPPPZZZATAAG-3΄) in water over a period of 50 μs and calculated DNA local parameters, step parameters, helix parameters, and major/minor groove widths to examine how the presence of multiple, consecutive nucleobase pairs might impact helical structure. In these simulations, the -containing DNA duplex exhibits a significantly wider major groove and greater average values of stagger, slide, rise, twist and h-rise than observed for a 'control' oligonucleotide in which nucleobase pairs are replaced by . The molecular origins of these structural changes are likely associated with at least two differences between and . First, the electrostatic properties of differ from in terms of density distribution and dipole moment. Second, differences are seen in the base stacking of pairs in dinucleotide steps, arising from energetically favorable stacking of the nitro group in with π-electrons of the adjacent base.
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    DNA Damage On The DOCK In FLT3-ITD-Driven Acute Myeloid Leukemia
    (Ferrata Storti Foundation, 2019-12) Pandey, Ruchi; Kapur, Reuben; Pediatrics, School of Medicine
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    DNA damage response protects against progressive kidney disease
    (American Society for Clinical Investigation, 2019-10) Molitoris, Bruce A.; Medicine, School of Medicine
    he pathophysiology of cellular injury and repair has been extensively studied in acute kidney injury (AKI) for more than 70 years. Although a great deal of knowledge has been generated, a debate over the importance of repairing damaged cells versus replacing them by proliferation remains. In this issue of the JCI, Kishi et al. demonstrate that following kidney epithelial cell injury, DNA repair, rather than cell proliferation, plays the central role in recovery and longevity by minimizing apoptosis, G2/M cell-cycle arrest, and subsequent fibrosis. This has important therapeutic implications and highlights the need for more sensitive techniques to evaluate functional, structural, and molecular recovery following injury.
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