Page 125 - 2014 Printable Abstract Book
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characterization and use of these two facilities will be presented and discussed.
(PS1-41) Role of various DNA repair pathways in chromosomal inversion formation in cho mutants.
Ian M. Cartwright and Takamitsu Kato, Colorado State University, Fort Collins, CO
Chromosomal inversions are considered to be stable chromosomal aberrations. Traditionally,
rBanding or gBanding techniques have been used to identify chromosomal inversions. The newest
technique being used to look at inversions is mBanding, this fluorescence technique limits the study to a
single chromosome and can only identify chromosomal inversion larger than 20 megabases. In this study
we have used a single-cycle EdU staining technique that uses an Alexa Fluor Azide fluorescent probe to
identify a single strand of DNA. Our protocol allows us to identify chromosomal inversions on the order of
0.6 megabases. In this study we have evaluated the role of various DNA repair pathways on the formation
of chromosomal inversions. Homologous recombination, nonhomologous endjoing, and fanconi anemia
CHO mutants were exposed to 0, 1, or 2 Gy of gamma irradiation and analyzed for inversions. To exclude
the possibility of false inversion attributed to two sister chromatid exchanges occurring on a single
chromatid we only counted inversions smaller than a width of a single chromatid, roughly 15 megabases
in size, which we classified as micro inversion. Using previous research, we confirmed that the observed
inversions were true inversions by comparing the number of observed inversions to the number of
observed rings at each dose. We used a Poisson distribution to calculate the expected “false” inversions,
2 sister chromatid exchange (SCE) on one chromosome. The values for total false inversions and 2 SCE
events within 15 megabases were similar to the observed numbers. We have shown that homologous
recombination repair is the primary repair pathway associated with inversion formation. The inversions
frequency in the mutant cells returns to the frequency observed in the control CHO10B2 when the
mutation is corrected.
(PS1-42) Alternative NHEJ pathway mediates inheritable DNA damage processing. Huichen Wang;
Tiedong Wang; Xiaobing Tang; and Ramesh Kandimalla, Emory University School of Medicine, Atlanta, GA
High linear energy transfer radiation induces clustered DNA damage including oxidized base
damage, DNA single-strand breaks and double-strand breaks around radiation tracks by direct and indirect
radiation effects. The mechanism of clustered DNA damage processing remains elusive. Previous data has
shown that poly(ADP-ribose) polymerase 1 (PARP-1) is involved in clustered DNA damage repair as a back-
up pathway of classical non homologous end joining (NHEJ) that depends on cell cycle. Here we
investigated the processing of clustered DNA damaged induced by heavy ions using live imaging DNA
damage track with fluorescent tagged DNA repair by coexpressing GFP-53BP1 and mCherryH2B), GFP-
Rad51 and mCherry-53BP1, as well as GFP-Mre11 and mCherry-53BP1 in cells 56Fe (1Gev/n). We found
that delayed 53BP foci tracks were distributed to progeny cells during mitosis, while all these proteins
formed DNA damage tracks. Unrepaired 53BP foci were duplicated post replication and equally
distributed into progeny cells after mitosis. Deletion or inhibition of PARP-1 increased the inheritable DNA
damage. CDK5 inhibitor reduced persistent DNA damage. Interaction of PARP-1, Mre11 and Rad51 on
inheritable DNA damage will be discussed. This work is supported by NASA space radiation program
NNX08BA08G.
123 | P a g e
(PS1-41) Role of various DNA repair pathways in chromosomal inversion formation in cho mutants.
Ian M. Cartwright and Takamitsu Kato, Colorado State University, Fort Collins, CO
Chromosomal inversions are considered to be stable chromosomal aberrations. Traditionally,
rBanding or gBanding techniques have been used to identify chromosomal inversions. The newest
technique being used to look at inversions is mBanding, this fluorescence technique limits the study to a
single chromosome and can only identify chromosomal inversion larger than 20 megabases. In this study
we have used a single-cycle EdU staining technique that uses an Alexa Fluor Azide fluorescent probe to
identify a single strand of DNA. Our protocol allows us to identify chromosomal inversions on the order of
0.6 megabases. In this study we have evaluated the role of various DNA repair pathways on the formation
of chromosomal inversions. Homologous recombination, nonhomologous endjoing, and fanconi anemia
CHO mutants were exposed to 0, 1, or 2 Gy of gamma irradiation and analyzed for inversions. To exclude
the possibility of false inversion attributed to two sister chromatid exchanges occurring on a single
chromatid we only counted inversions smaller than a width of a single chromatid, roughly 15 megabases
in size, which we classified as micro inversion. Using previous research, we confirmed that the observed
inversions were true inversions by comparing the number of observed inversions to the number of
observed rings at each dose. We used a Poisson distribution to calculate the expected “false” inversions,
2 sister chromatid exchange (SCE) on one chromosome. The values for total false inversions and 2 SCE
events within 15 megabases were similar to the observed numbers. We have shown that homologous
recombination repair is the primary repair pathway associated with inversion formation. The inversions
frequency in the mutant cells returns to the frequency observed in the control CHO10B2 when the
mutation is corrected.
(PS1-42) Alternative NHEJ pathway mediates inheritable DNA damage processing. Huichen Wang;
Tiedong Wang; Xiaobing Tang; and Ramesh Kandimalla, Emory University School of Medicine, Atlanta, GA
High linear energy transfer radiation induces clustered DNA damage including oxidized base
damage, DNA single-strand breaks and double-strand breaks around radiation tracks by direct and indirect
radiation effects. The mechanism of clustered DNA damage processing remains elusive. Previous data has
shown that poly(ADP-ribose) polymerase 1 (PARP-1) is involved in clustered DNA damage repair as a back-
up pathway of classical non homologous end joining (NHEJ) that depends on cell cycle. Here we
investigated the processing of clustered DNA damaged induced by heavy ions using live imaging DNA
damage track with fluorescent tagged DNA repair by coexpressing GFP-53BP1 and mCherryH2B), GFP-
Rad51 and mCherry-53BP1, as well as GFP-Mre11 and mCherry-53BP1 in cells 56Fe (1Gev/n). We found
that delayed 53BP foci tracks were distributed to progeny cells during mitosis, while all these proteins
formed DNA damage tracks. Unrepaired 53BP foci were duplicated post replication and equally
distributed into progeny cells after mitosis. Deletion or inhibition of PARP-1 increased the inheritable DNA
damage. CDK5 inhibitor reduced persistent DNA damage. Interaction of PARP-1, Mre11 and Rad51 on
inheritable DNA damage will be discussed. This work is supported by NASA space radiation program
NNX08BA08G.
123 | P a g e
