Page 105 - 2014 Printable Abstract Book
P. 105
PS1 DNA DAMAGE, DNA REPAIR, CELL SIGNALING
(PS1-01) Low energy electrons induce loss of plasmid functionality by producing lethal lesions in DNA.
Saloua Kouass Sahbani; Pierre Cloutier; Andrew Bass; Leon Sanche; and Darel J Hunting, université de
sherbrooke, sherbrooke, Canada
Secondary electrons are the most abundant species generated by the interaction of ionizing
radiation with biological matter and most of them have low energies. These low energy electrons (LEEs)
have been experimentally found to induce DNA damage, including base damage, base release, and strand
breaks; however, their cellular lethality has never been directly demonstrated. We used a simple model
system comprising E. coli1 transformed with a LEE-irradiated plasmid, to investigate the effect of LEEs on
DNA functionality. The DNA films were prepared with a molecular self-assembly technique2, in which the
DNA is condensed onto a pyrolitic graphite substrate in the presence of 1, 3-diaminopropane molecules.
The assembly mimics the amino groups of the lysine and arginine amino acids within the histone proteins
in nucleosomes. These films, which have a high uniformity compared to those prepared by lyophilization,
were irradiated with 10 eV LEEs. The quantities of plasmids rendered non-functional depended on the
electron fluence. The data were correlated to LEE-induced single strand breaks (SSBs), isolated base
damage, frank double-strand breaks (DSB) and cluster damage. The latter was revealed by digestion with
the repair enzymes, Nth and/or Fpg followed by gel electrophoresis to quantify additional SSB and DSB.
SSBs and isolated base damage are known to be readily repaired and thus relatively non-toxic. DSBs which
are known to be the most toxic lesion, had their levels too low to explain the loss of plasmid functionality
observed after irradiation with LEEs. For example, when 10 % and 38 % of the plasmids were rendered
non-functional only 1.1 % and 2.6 %, respectively, had a DSB. In addition, occult DSBs, revealed by
digestion with repair enzymes, were also formed relatively infrequently such that when 10 % and 38 % of
the plasmids were rendered non-functional, only 3.8 % and 6.8 %, respectively, had occult DSBs, a type of
cluster lesion. We conclude that LEEs induce several types of lethal lesions, including Frank DSBs and
cluster damage, but the majority of the lethal lesions are not yet identified and may be either DNA
interstrand crosslinks or DNA-diaminopropane adducts. 1-Kouass Sahbani S., et al. Radiat. Res. (2013). 2-
Boulanouar O, et al. The Journal of Physical Chemistry C 115: 2191. (2011).
(PS1-04) Cisplatin Adducts Sensitize DNA to Attack by Hydrated Electrons Leading to Cisplatin
Detachment and Base Damage. Behnaz Behmand, MSc; Pierre Cloutier, MSc; Richard Wagner, Pr; Leon
Sanche, Pr; Darel Hunting, Pr; Jean-Louis Marignier, Pr; and Mehran Mostafavi, Pr, Université de
Sherbrooke, Sherbrooke, Canada
Cisplatin [PtCl2 (NH3)2] is a chemotherapeutic agent that chemically binds to DNA purines bases,
especially guanine (G) at position N7 [1]. Recent experiments have shown enhancement of single and
double strand breaks by low energy electrons in dry supercoiled plasmid containing low levels of cisplatin
adducts [2]. In the present work, we studied hydrated electron interactions with short oligonucleotides
(5'-TTTTTGTGTTT-3' and 5'-TTTTTTTGTTT-3') containing cisplatin adducts on the guanine(s). Cisplatin-
oligonucleotides, with either a cisplatin monoadduct or a diadduct between the guanines were labelled
with 32P and exposed to hydrated electrons in solution generated by gamma ray induced radiolysis.
103 | P a g e
(PS1-01) Low energy electrons induce loss of plasmid functionality by producing lethal lesions in DNA.
Saloua Kouass Sahbani; Pierre Cloutier; Andrew Bass; Leon Sanche; and Darel J Hunting, université de
sherbrooke, sherbrooke, Canada
Secondary electrons are the most abundant species generated by the interaction of ionizing
radiation with biological matter and most of them have low energies. These low energy electrons (LEEs)
have been experimentally found to induce DNA damage, including base damage, base release, and strand
breaks; however, their cellular lethality has never been directly demonstrated. We used a simple model
system comprising E. coli1 transformed with a LEE-irradiated plasmid, to investigate the effect of LEEs on
DNA functionality. The DNA films were prepared with a molecular self-assembly technique2, in which the
DNA is condensed onto a pyrolitic graphite substrate in the presence of 1, 3-diaminopropane molecules.
The assembly mimics the amino groups of the lysine and arginine amino acids within the histone proteins
in nucleosomes. These films, which have a high uniformity compared to those prepared by lyophilization,
were irradiated with 10 eV LEEs. The quantities of plasmids rendered non-functional depended on the
electron fluence. The data were correlated to LEE-induced single strand breaks (SSBs), isolated base
damage, frank double-strand breaks (DSB) and cluster damage. The latter was revealed by digestion with
the repair enzymes, Nth and/or Fpg followed by gel electrophoresis to quantify additional SSB and DSB.
SSBs and isolated base damage are known to be readily repaired and thus relatively non-toxic. DSBs which
are known to be the most toxic lesion, had their levels too low to explain the loss of plasmid functionality
observed after irradiation with LEEs. For example, when 10 % and 38 % of the plasmids were rendered
non-functional only 1.1 % and 2.6 %, respectively, had a DSB. In addition, occult DSBs, revealed by
digestion with repair enzymes, were also formed relatively infrequently such that when 10 % and 38 % of
the plasmids were rendered non-functional, only 3.8 % and 6.8 %, respectively, had occult DSBs, a type of
cluster lesion. We conclude that LEEs induce several types of lethal lesions, including Frank DSBs and
cluster damage, but the majority of the lethal lesions are not yet identified and may be either DNA
interstrand crosslinks or DNA-diaminopropane adducts. 1-Kouass Sahbani S., et al. Radiat. Res. (2013). 2-
Boulanouar O, et al. The Journal of Physical Chemistry C 115: 2191. (2011).
(PS1-04) Cisplatin Adducts Sensitize DNA to Attack by Hydrated Electrons Leading to Cisplatin
Detachment and Base Damage. Behnaz Behmand, MSc; Pierre Cloutier, MSc; Richard Wagner, Pr; Leon
Sanche, Pr; Darel Hunting, Pr; Jean-Louis Marignier, Pr; and Mehran Mostafavi, Pr, Université de
Sherbrooke, Sherbrooke, Canada
Cisplatin [PtCl2 (NH3)2] is a chemotherapeutic agent that chemically binds to DNA purines bases,
especially guanine (G) at position N7 [1]. Recent experiments have shown enhancement of single and
double strand breaks by low energy electrons in dry supercoiled plasmid containing low levels of cisplatin
adducts [2]. In the present work, we studied hydrated electron interactions with short oligonucleotides
(5'-TTTTTGTGTTT-3' and 5'-TTTTTTTGTTT-3') containing cisplatin adducts on the guanine(s). Cisplatin-
oligonucleotides, with either a cisplatin monoadduct or a diadduct between the guanines were labelled
with 32P and exposed to hydrated electrons in solution generated by gamma ray induced radiolysis.
103 | P a g e
