Page 34 - 2014 Printable Abstract Book
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not affect the HRR efficiency in cells using a modified pDR-GFP-I-SceI system. Taken together, our results
reveal that besides high-LET radiation directly inducing small DNA fragments; Ape1 enzyme digestion also
contributes to the generation of small DNA fragments in high-LET irradiated cells and contributes to the
RBE on cell killing.
(S203) Expression of bacterial Ku in eukaryotes to disrupt double strand break repair. Kanchanjunga
Prasai; Reneau Castore; David M. Krzywanski, PhD; Lucy C. Robinson, PhD; Kelly Tatchell, Ph.D; Lynn
Harrison, LSU Health Sciences Center at Shreveport, Shreveport, LA
The aim of this work is to generate a tool to disrupt DNA double strand break (DSB) repair in the
nucleus or the mitochondria. It is well-documented that disruption of DSB repair in the nucleus sensitizes
cells to ionizing radiation. Little is known about mitochondrial DSB repair. A functional mitochondrial
electron transport chain (ETC) is required for normal cell function, as well as cancer cell viability. Since
mitochondrial DNA (mtDNA) encodes ETC proteins, we hypothesized that interference with mtDNA repair
after irradiation could inhibit growth of cancer cells and enhance killing by production of reactive oxygen
species from dysfunctional mitochondria. We have generated fusion proteins of Mycobacterium
tuberculosis or Mycobacterium marinum Ku that are targeted either to the nucleus or the mitochondria.
Nuclear-targeted bacterial Ku in human cells binds sites of laser-induced DNA damage, inhibits
homologous recombination and sensitizes cells to bleomycin sulfate. To test whether bacterial Ku disrupts
mitochondrial function, we expressed the mitochondrial-targeted bacterial Ku in yeast. The yeast model
was chosen as there is an easy test for functional mitochondria: yeast can grow on glucose but not on a
non-fermentable carbon source if the ETC is not functioning. We determined that expression of the
bacterial Ku in mitochondria decreases functional ETC and decreases the mtDNA copy number. Loss of
mtDNA under normal growing conditions is likely related to inhibition of DNA replication. Using a ChIP-
type assay, we have demonstrated that bacterial Ku binds to a greater extent to the origin of replication
5 than to the DNA that encodes COX1. To test whether bacterial Ku can disrupt the mitochondrial function
in human cells, the mitochondrial-targeted bacterial Ku was expressed in MCF7 cells. The bacterial Ku
does not however alter growth rate or sensitivity to DNA damage in human cells even though the bacterial
Ku does bind mtDNA. It is possible that different mechanisms of mtDNA replication in yeast and human
cells account for the different biological effects of bacterial Ku in the two eukaryotes. Using expression of
mitochondrial-targeted Xho I restriction enzyme, we hope to understand whether the bacterial Ku can
block DSB repair in eukaryote mitochondria and whether mtDNA repair is important for cell survival.
(S204) Polynucleotide kinase/phosphatase, PNKP, as a target for enhanced cancer therapy. Zahra Shire,
University of Alberta, Edmonton, Canada
Polynucleotide kinase/phosphatase, PNKP, as a target for enhanced cancer therapy
the radioresistance and chemoresistance of tumors are major obstacles that may lead to the failure of
cancer therapy. Reducing DNA repair in cancer cells is emerging as a new strategy for improving cancer
treatment. One approach is to identify small molecules inhibitors of DNA repair enzymes. Human
polynucleotide kinase/phosphatase (hPNKP), which possesses DNA 5’-kinase and 3’-phosphatase
activities, plays an essential role in DNA strand break repair by rendering strand-break termini suitable for
DNA polymerases and ligases. Previous work in our lab has shown that reduced expression of PNKP
32 | P a g e
reveal that besides high-LET radiation directly inducing small DNA fragments; Ape1 enzyme digestion also
contributes to the generation of small DNA fragments in high-LET irradiated cells and contributes to the
RBE on cell killing.
(S203) Expression of bacterial Ku in eukaryotes to disrupt double strand break repair. Kanchanjunga
Prasai; Reneau Castore; David M. Krzywanski, PhD; Lucy C. Robinson, PhD; Kelly Tatchell, Ph.D; Lynn
Harrison, LSU Health Sciences Center at Shreveport, Shreveport, LA
The aim of this work is to generate a tool to disrupt DNA double strand break (DSB) repair in the
nucleus or the mitochondria. It is well-documented that disruption of DSB repair in the nucleus sensitizes
cells to ionizing radiation. Little is known about mitochondrial DSB repair. A functional mitochondrial
electron transport chain (ETC) is required for normal cell function, as well as cancer cell viability. Since
mitochondrial DNA (mtDNA) encodes ETC proteins, we hypothesized that interference with mtDNA repair
after irradiation could inhibit growth of cancer cells and enhance killing by production of reactive oxygen
species from dysfunctional mitochondria. We have generated fusion proteins of Mycobacterium
tuberculosis or Mycobacterium marinum Ku that are targeted either to the nucleus or the mitochondria.
Nuclear-targeted bacterial Ku in human cells binds sites of laser-induced DNA damage, inhibits
homologous recombination and sensitizes cells to bleomycin sulfate. To test whether bacterial Ku disrupts
mitochondrial function, we expressed the mitochondrial-targeted bacterial Ku in yeast. The yeast model
was chosen as there is an easy test for functional mitochondria: yeast can grow on glucose but not on a
non-fermentable carbon source if the ETC is not functioning. We determined that expression of the
bacterial Ku in mitochondria decreases functional ETC and decreases the mtDNA copy number. Loss of
mtDNA under normal growing conditions is likely related to inhibition of DNA replication. Using a ChIP-
type assay, we have demonstrated that bacterial Ku binds to a greater extent to the origin of replication
5 than to the DNA that encodes COX1. To test whether bacterial Ku can disrupt the mitochondrial function
in human cells, the mitochondrial-targeted bacterial Ku was expressed in MCF7 cells. The bacterial Ku
does not however alter growth rate or sensitivity to DNA damage in human cells even though the bacterial
Ku does bind mtDNA. It is possible that different mechanisms of mtDNA replication in yeast and human
cells account for the different biological effects of bacterial Ku in the two eukaryotes. Using expression of
mitochondrial-targeted Xho I restriction enzyme, we hope to understand whether the bacterial Ku can
block DSB repair in eukaryote mitochondria and whether mtDNA repair is important for cell survival.
(S204) Polynucleotide kinase/phosphatase, PNKP, as a target for enhanced cancer therapy. Zahra Shire,
University of Alberta, Edmonton, Canada
Polynucleotide kinase/phosphatase, PNKP, as a target for enhanced cancer therapy
the radioresistance and chemoresistance of tumors are major obstacles that may lead to the failure of
cancer therapy. Reducing DNA repair in cancer cells is emerging as a new strategy for improving cancer
treatment. One approach is to identify small molecules inhibitors of DNA repair enzymes. Human
polynucleotide kinase/phosphatase (hPNKP), which possesses DNA 5’-kinase and 3’-phosphatase
activities, plays an essential role in DNA strand break repair by rendering strand-break termini suitable for
DNA polymerases and ligases. Previous work in our lab has shown that reduced expression of PNKP
32 | P a g e
