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You searched for +publisher:"Virginia Commonwealth University" +contributor:("Dr. Lawrence F. Povirk"). Showing records 1 – 3 of 3 total matches.

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Virginia Commonwealth University

1. Kawale, Ajinkya S. Interplay between Artemis and TDP1 in sensitivity to radiomimetic agents.

Degree: MS, Pharmacology & Toxicology, 2015, Virginia Commonwealth University

DNA double-strand breaks containing unligatable termini are potent cytotoxic lesions leading to cell death or growth arrest. Artemis, which is associated with the Non-Homologous End Joining (NHEJ) pathway, is the major end processing nuclease that resolves unligatable termini, especially the 3′ blocks, by nucleolytic trimming. Tyrosyl-DNA Phosphodiesterase 1 (TDP1) is an enzyme which is biochemically competent in 3′-phosphoglycolate processing. The purpose of this study is to investigate if TDP1 is an end-processing enzyme involved in the NHEJ pathway. Clonogenic Survival assays using shRNA-mediated TDP1 knockdown and Artemis knockout (Artemis-/-) in HCT116 cells showed increased sensitivity to Neocarzinostatin (NCS) and Calicheamicin, radiomimetic drugs that produce 3′-phosphoglycolate-terminated double-strand breaks. Thus, a cell line with combined deficiency in Artemis and TDP1 was generated by infecting Artemis-/- single mutants with a lentivirus expressing a TDP1 shRNA. Positive clones were screened for maximum TDP1 knockdown which was found to be 10X. Clonogenic survival assays carried out on shTDP1 & Artemis-/- single mutants and the Artemis-/-.shTDP1 double mutants showed similar sensitivity to Calicheamicin and NCS. Immunofluorescence studies on Art-/- and Art-/-.shTDP1 mutants also showed a similar increase in persistent 53BP1 foci, a measure of DNA damage, after treatment with NCS. Cell cycle analysis studies showed all these mutants arrest in G1 phase of the cell cycle after treatment with NCS. Thus, taken all together, surprisingly, these experiments suggest that TDP1 functions are epistatic with Artemis in the NHEJ pathway for repair of Calicheamicin- and NCS-mediated DNA damage. Advisors/Committee Members: Dr. Lawrence F. Povirk.

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APA (6th Edition):

Kawale, A. S. (2015). Interplay between Artemis and TDP1 in sensitivity to radiomimetic agents. (Thesis). Virginia Commonwealth University. Retrieved from https://doi.org/10.25772/Z955-1E26 ; https://scholarscompass.vcu.edu/etd/3755

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16th Edition):

Kawale, Ajinkya S. “Interplay between Artemis and TDP1 in sensitivity to radiomimetic agents.” 2015. Thesis, Virginia Commonwealth University. Accessed April 16, 2021. https://doi.org/10.25772/Z955-1E26 ; https://scholarscompass.vcu.edu/etd/3755.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7th Edition):

Kawale, Ajinkya S. “Interplay between Artemis and TDP1 in sensitivity to radiomimetic agents.” 2015. Web. 16 Apr 2021.

Vancouver:

Kawale AS. Interplay between Artemis and TDP1 in sensitivity to radiomimetic agents. [Internet] [Thesis]. Virginia Commonwealth University; 2015. [cited 2021 Apr 16]. Available from: https://doi.org/10.25772/Z955-1E26 ; https://scholarscompass.vcu.edu/etd/3755.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Kawale AS. Interplay between Artemis and TDP1 in sensitivity to radiomimetic agents. [Thesis]. Virginia Commonwealth University; 2015. Available from: https://doi.org/10.25772/Z955-1E26 ; https://scholarscompass.vcu.edu/etd/3755

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

2. Kawale, Ajinkya S. PROCESSING OF 3′-BLOCKED DNA DOUBLE-STRAND BREAKS BY TYROSYL-DNA PHOSPHODIESTERASE 1, ARTEMIS AND POLYNUCLEOTIDE KINASE/ PHOSPHATASE.

Degree: PhD, Pharmacology & Toxicology, 2018, Virginia Commonwealth University

DNA double-strand breaks (DSBs) containing unligatable termini are potent cytotoxic lesions leading to growth arrest or cell death. The Artemis nuclease and tyrosyl-DNA phosphodiesterase (TDP1) are each capable of resolving protruding 3′-phosphoglycolate (PG) termini of DNA double-strand breaks (DSBs). Consequently, a knockout of Artemis and a knockout/knockdown of TDP1 rendered cells sensitive to the radiomimetic agent neocarzinostatin (NCS), which induces 3′-PG-terminated DSBs. Unexpectedly, however, a knockdown or knockout of TDP1 in Artemis-null cells did not confer any greater sensitivity than either deficiency alone, indicating a strict epistasis between TDP1 and Artemis. Moreover, a deficiency in Artemis, but not TDP1, resulted in a fraction of unrepaired DSBs, which were assessed as 53BP1 foci. Conversely, a deficiency in TDP1, but not Artemis, resulted in a dramatic increase in dicentric chromosomes following NCS treatment. An inhibitor of DNA-dependent protein kinase, a key regulator of the classical nonhomologous end joining (C-NHEJ) pathway sensitized cells to NCS but eliminated the sensitizing effects of both TDP1 and Artemis deficiencies. Moreover, Polynucleotide Kinase/ Phosphatase (PNKP) is known to process 3′-phosphates and 5′-hydroxyls during DSB repair. PNKP-deficiency sensitized both HCT116 and HeLa cells to 3′-phosphate ended DSBs formed upon radiation and radiomimetic drug treatment. The increased cytotoxicity in the absence of PNKP was synonymous with persistent, un-rejoined 3′-phosphate-ended DSBs. However, DNA-PK deficiency sensitized PNKP-/- cells to low doses of NCS suggesting that, in the absence of PNKP, alternative enzyme(s) can remove 3′-phosphates in a DNA-PK-dependent manner. These results suggest that TDP1 and Artemis perform different functions in the repair of terminally blocked DSBs by the C-NHEJ pathway, and that whereas an Artemis deficiency prevents end joining of some DSBs, a TDP1 deficiency tends to promote DSB mis-joining. In addition, loss of PNKP significantly sensitizes cells to 3′-phosphate-ended DSBs due to a defect in 3′-dephosphorylation. Advisors/Committee Members: Dr. Lawrence F. Povirk.

Subjects/Keywords: TDP1; Artemis; Epistasis; Double-strand break repair; NHEJ; Biochemistry; Molecular Biology; Molecular Genetics

…2018. Advisor: Dr. Lawrence F. Povirk, Professor, Department of Pharmacology and Toxicology… …Doctor of Philosophy at Virginia Commonwealth University. Virginia Commonwealth University… 

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Kawale, A. S. (2018). PROCESSING OF 3′-BLOCKED DNA DOUBLE-STRAND BREAKS BY TYROSYL-DNA PHOSPHODIESTERASE 1, ARTEMIS AND POLYNUCLEOTIDE KINASE/ PHOSPHATASE. (Doctoral Dissertation). Virginia Commonwealth University. Retrieved from https://doi.org/10.25772/HX1V-JT51 ; https://scholarscompass.vcu.edu/etd/5329

Chicago Manual of Style (16th Edition):

Kawale, Ajinkya S. “PROCESSING OF 3′-BLOCKED DNA DOUBLE-STRAND BREAKS BY TYROSYL-DNA PHOSPHODIESTERASE 1, ARTEMIS AND POLYNUCLEOTIDE KINASE/ PHOSPHATASE.” 2018. Doctoral Dissertation, Virginia Commonwealth University. Accessed April 16, 2021. https://doi.org/10.25772/HX1V-JT51 ; https://scholarscompass.vcu.edu/etd/5329.

MLA Handbook (7th Edition):

Kawale, Ajinkya S. “PROCESSING OF 3′-BLOCKED DNA DOUBLE-STRAND BREAKS BY TYROSYL-DNA PHOSPHODIESTERASE 1, ARTEMIS AND POLYNUCLEOTIDE KINASE/ PHOSPHATASE.” 2018. Web. 16 Apr 2021.

Vancouver:

Kawale AS. PROCESSING OF 3′-BLOCKED DNA DOUBLE-STRAND BREAKS BY TYROSYL-DNA PHOSPHODIESTERASE 1, ARTEMIS AND POLYNUCLEOTIDE KINASE/ PHOSPHATASE. [Internet] [Doctoral dissertation]. Virginia Commonwealth University; 2018. [cited 2021 Apr 16]. Available from: https://doi.org/10.25772/HX1V-JT51 ; https://scholarscompass.vcu.edu/etd/5329.

Council of Science Editors:

Kawale AS. PROCESSING OF 3′-BLOCKED DNA DOUBLE-STRAND BREAKS BY TYROSYL-DNA PHOSPHODIESTERASE 1, ARTEMIS AND POLYNUCLEOTIDE KINASE/ PHOSPHATASE. [Doctoral Dissertation]. Virginia Commonwealth University; 2018. Available from: https://doi.org/10.25772/HX1V-JT51 ; https://scholarscompass.vcu.edu/etd/5329


Virginia Commonwealth University

3. Tatavarthi, Haritha. Action of Tyrosyl DNA Phosphodiesterase on 3'-Phosphoglycolate Terminated DNA Strand Breaks.

Degree: MS, Pharmacology & Toxicology, 2006, Virginia Commonwealth University

Free radical-mediated DNA double strand breaks (DSBs) are induced either directly by ionizing radiation or by certain chemicals like bleomycin. These breaks are terminated by 3'-PG (PO4CH2COOˉ) or 3'-phosphate groups formed as a result of fragmentation of deoxyribose. To study the nature of repair of these 3'-blocked breaks, we constructed substrates mimicking free-radical induced DSBs. Human and yeast tyrosyl DNA-phosphodiesterase (Tdpl) efficiently processed substrates with 3'-PGs, in either the presence or absence of magnesium, to give a 3'-phosphate. Gel filtration chromatography and western blotting codmed that the putative enzyme in human extracts that efficiently processed PG was indeed tyrosyl DNA-phosphodiesterase. When recombinant hTdpl was purified using HiTrap nickel chelating columns and its PG processing activity compared to that of partially purified native enzyme (from lymphoblastoid whole-cell extracts using Sephacryl S-300 gel filtration columns), we found that the recombinant enzyme had lesser 3'-PG removal activity than the partially purified native enzyme. On cloning recombinant FLAG-tagged hTdpl into human expression vectors, we observed that the FLAG epitope tag did not show any evidence of affecting the specificity of the enzyme. Due to the many differences between bacterial and human cells, we cloned recombinant FLAG-tagged hTdpl into U-87 cells (adenovirus infected glioma cell) and this recombinant enzyme showed the same specificity toward PG substrates as when prepared from bacteria. End-processing assays using the NHEJ proteins- Ku, DNA-PK and XRCC4/Ligase IV-alone or in combination showed an inhibition of hTdpl activity on 3'- overhangs. In nuclear extracts, hTdp1 association with XRCC1, a single-strand repair protein, showed to increase the PG-processing activity of Tdpl up to 4 times. Whole-cell extracts containing mutant Tdpl derived from patients suffering from spinocerebellar axonal neuropathy (SCAN1) were found to be deficient in PG-processing. Addition of JRLl whole-cell extract (SCAN1 extract containing mutant Tdpl) to purified FLAG-tagged hTdpl showed to decrease the phosphotyrosyl processing and increase the PG-processing of FLAG-tagged hTdpl suggesting that there must be other factors in the extract that affect the enzyme activity. Experiments carried out to check for the presence of Tdpl in mitochondrial extracts obtained from GM1310 normal human fibroblasts as well as in SCANl (JRL) mitochondrial extracts, showed that mitochondrial extracts contained Tdpl at a concentration comparable to whole-cell extracts. Our results also showed that mitochondrial extracts from the SCANl cell-line, JRL3 (containing mutant Tdpl), lacked detectable Tdpl activity suggesting that all PG-processing activity in mitochondria may be attributable to Tdpl. Advisors/Committee Members: Dr. Lawrence F. Povirk.

Subjects/Keywords: Sephacryl; XRCC4/Ligase IV; FLAG-tagged hTdpl; mitochondria; Medical Pharmacology; Medical Sciences; Medicine and Health Sciences

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Tatavarthi, H. (2006). Action of Tyrosyl DNA Phosphodiesterase on 3'-Phosphoglycolate Terminated DNA Strand Breaks. (Thesis). Virginia Commonwealth University. Retrieved from https://doi.org/10.25772/6Q3X-5718 ; https://scholarscompass.vcu.edu/etd/1140

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16th Edition):

Tatavarthi, Haritha. “Action of Tyrosyl DNA Phosphodiesterase on 3'-Phosphoglycolate Terminated DNA Strand Breaks.” 2006. Thesis, Virginia Commonwealth University. Accessed April 16, 2021. https://doi.org/10.25772/6Q3X-5718 ; https://scholarscompass.vcu.edu/etd/1140.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7th Edition):

Tatavarthi, Haritha. “Action of Tyrosyl DNA Phosphodiesterase on 3'-Phosphoglycolate Terminated DNA Strand Breaks.” 2006. Web. 16 Apr 2021.

Vancouver:

Tatavarthi H. Action of Tyrosyl DNA Phosphodiesterase on 3'-Phosphoglycolate Terminated DNA Strand Breaks. [Internet] [Thesis]. Virginia Commonwealth University; 2006. [cited 2021 Apr 16]. Available from: https://doi.org/10.25772/6Q3X-5718 ; https://scholarscompass.vcu.edu/etd/1140.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Tatavarthi H. Action of Tyrosyl DNA Phosphodiesterase on 3'-Phosphoglycolate Terminated DNA Strand Breaks. [Thesis]. Virginia Commonwealth University; 2006. Available from: https://doi.org/10.25772/6Q3X-5718 ; https://scholarscompass.vcu.edu/etd/1140

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

.