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Title PROCESSING OF 3′-BLOCKED DNA DOUBLE-STRAND BREAKS BY TYROSYL-DNA PHOSPHODIESTERASE 1, ARTEMIS AND POLYNUCLEOTIDE KINASE/ PHOSPHATASE
URL
Publication Date
Degree PhD
Discipline/Department Pharmacology & Toxicology
Degree Level doctoral
University/Publisher Virginia Commonwealth University
Abstract 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<sup>-/-</sup> 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.
Subjects/Keywords TDP1; Artemis; Epistasis; Double-strand break repair; NHEJ; Biochemistry; Molecular Biology; Molecular Genetics
Contributors Dr. Lawrence F. Povirk
Country of Publication us
Record ID oai:scholarscompass.vcu.edu:etd-6432
Repository vcu
Date Retrieved
Date Indexed 2020-07-20
Created Date 2018-01-01 08:00:00

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…curve assay ............................................................................................................. 51 3.4 TDP1 activity assay…

…57 vii 4. RESULTS ............................................................................................................................. 59 4.1 Generation of TDP1-mutant (shTDP1 and TDP1-/-) cell lines…

…59 4.2 TDP1 deficient cells are hypersensitive to ionizing radiation and radiomimetic agents. ... 63 4.3 Generation of TDP1 and Artemis double knockouts .......................................................... 66 4.4 TDP1 and Artemis are epistatic…

…for the repair of 3′-PG DSBs via NHEJ ........................... 71 4.5 Absence of Artemis but not TDP1 confers a defect in DSB rejoining ............................... 77 4.6 Loss of TDP1 but not Artemis leads to misjoining of 3′-PG DSBs…

…85 4.7 Absence of Artemis delays G1-S progression upon DSB induction; this delay is rescued in an additional absence of TDP1.................................................................................................. 91 4.8 Interplay between…

TDP1/Artemis with the DNA damage response proteins PARP1 and ATM. ......................................................................................................................................... 96 4.9 PNKP-deficient cells display enhanced…

…21 Figure 1-6: Mechanism of NCS action and typical DSB ends formed ......................................... 22 Figure 1-7: NCS and CAL induced bistranded lesions with specific modifications .................... 23 Figure 2-1: Structure of TDP1

…28 Figure 2-2: Catalytic mechanism of TDP1 in its canonical function ............................................ 29 Figure 3-1: Lentiviral transfer vector pLSLPw harboring the shRNA against TDP1 .................. 50 Figure 4-1- TDP1 expression in…

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