The effects of microgravity on ligase activity in DNA repair of DSB during space flight

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The effects of microgravity on ligase activity in
DNA repair of DSB during space flight Shunji
Nagaoka1, Akihisa Takahashi2, Ken Ohnishi2,
Tamotsu Nakano3 and Takeo Ohnishi2 1Department
of Gravitational Physiology, Fujita Health
University School of Health Sciences, Toyoake,
Aichi, 470-1192, 2Department of Biology, Nara
Medical University, Kashihara, Nara, 634-8521,
3Space Experiment Department, National Space
Development Agency (NASDA) of Japan, Tsukuba,
Ibaraki, 305-0047, Japan.
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Summary
In recent years, some contradictory data about
the effects of microgravity on radiation-induced
biological responses in space experiments have
been reported. The aim of the present study was
to clarify whether enzymatic repair of DNA
double-strand breaks is affected by microgravity
using an in vitro enzymatic reaction system. We
measured the DNA repair activity in vitro of T4
DNA ligase as a DNA substrate damaged by
restriction enzyme digestion under microgravity
(Discovery STS-91). After the flight, the amount
of ligated DNA molecules was measured using
electrophoresis methods. In the space sample, the
ligated products were produced by T4 DNA ligase
activity for double-strand break DNA, and
increased with increasing T4 DNA ligase
concentration. We detected almost no difference
in T4 DNA ligase activity between the space
experiments and the control ground experiments.
No significant effect of microgravity on ligation
of damaged DNA was found during space flight.
Therefore, other mechanisms must account for the
synergism between radiation and microgravity, if
it exists.
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Background
In recent years, some contradictory data about
the effects of microgravity on radiation-induced
biological responses in space experiments have
been reported.
(?) enhancement abnormal development
mutation frequency (?) decrease
radiosensitivity (?) no effect mutation
frequency repair activity
C. morosus Bucker et al. 1986
D. melanogaster Ikenaga et al. 1997
D. radiodurans Kobayashi et al. 1996
E. coli Harada et al. 1996
D. discoideum Ohnishi et al. 1997
S. cerevisiae Kiefer et al. 1999
E. coli Horneck et al. 1996
human cells Horneck et al. 1996
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Question
Whether is ligation step affected by microgravity
against DNA strand break?
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Purpose
The aim of this study was to clarify whether
enzymatic repair of DNA double-strand breaks is
affected by microgravity using an in vitro
enzymatic reaction system.
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Preliminary experiment on ground
We performed very simple experiments to determine
the possible effect of microgravity on repair of
DSBs. We studied the DSBs produced using
restriction endonuclease, because the kinetics of
the rejoining of restriction endonuclease-induced
DSBs are similar to those of radiation-induced
DSBs (Cheong and Iliakis 1997). We chosen SmaI
restriction endonuclease, because the ligation of
the SmaI site was slower than that of cohesive
end sites and of the blunt ended sites of HaeIII,
AluI and HincII (personal communication by TOYOBO
Co., Ltd. and TaKaRa Shuzo Co., Ltd.). To
determine suitable reaction times for T4 DNA
ligase activity in the refrigerator and in the
Space Shuttles cabin, we analyzed the rejoining
of DSBs of plasmid DNA after treatment with T4
DNA ligase at 4 or 22.5C for 0-20 h by agarose
gel electrophoresis.
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Result 1
Although the DNA rejoining reaction reached
saturation after 12 h, a linear time-course was
observed from 0-12 h at 22.5C with high ligase
concentration (1.0 U/µg). At low temperature
(4C) or with low ligase concentration (0.3
U/µg), the DNA rejoining reaction was distinctly
slower and linear until 20 h.
1.0 U/?g
0.3 U/?g
M
2
4
8
12
20
L
U
M
2
4
8
12
20
L
U
multimeric Ligated products
22.5oC
ocDNA
linear DNA
ccDNA
multimeric Ligated products
4oC
ocDNA
linear DNA
ccDNA
Fig. 1. T4 DNA ligase activity at 4 or 22.5C on
ground. Typical pattern after gel
electrophoresis. Lane U, untreated pRC lane L,
pRC digested by Sma I lane M, molecular markers
(?DNA-Hind III fragments). 2, 4, 8, 12 and 20,
reaction time (h) with 0.3 or 1.0 U/?g of T4 DNA
ligase at 4 or 22.5C.
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Result 2
From these data, we determined the appropriate T4
DNA ligase concentrations for 10 h of incubation
at the Space Shuttles cabin temperature (22.5C)
or 15 h in the refrigerator (4C) to achieve a
linear rejoining reaction.
100
75
50
Relative amounts ()
25
0
Incubation time (h)
Fig. 2. T4 DNA ligase activity at 4 or 22.5C on
ground. Relative amount of ligated products
(ccDNA, ocDNA and multimeric ligated products).
Closed symbols, with 1.0 U/?g of T4 DNA ligase
open symbols, with 0.3 U/?g of T4 DNA lieges.
Circles, at 4C triangles, at 22.5C.
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Janet Lynn Kavadi (Mission specialist)
Act.
Deact.
2/1417 3/1629
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Experimental outline
plasmid DNA (pRC) ? restriction enzyme Sma I
alkylated DNA (5µg) 10µl of polymerase
200µl of substrate ? mix
(activation) incubation at ? 25 h in room
temperature (SC) 300µl of stop
solution ?
mix (deactivation) ?
freeze ? preparation by
Phenol ? precipitation
by ethanol ? mutation assay
launch
in Space
landing
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A new experimental equipment
In space, it is difficult to take exact volume of
chemical solution and to mix chemical solution in
handling. Therefore, we developed a new equipment
for space experiments.
template DNA substrate
ligase
stop soln.
before activation
after activation
after deactivation
special temporary sealing band
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Result 3
Ligated products (ccDNA, ocDNA and multimeric
ligated products) were produced by ligase
treatment of linear DNA containing DSBs and
increased in a manner dependent on ligase
concentration (0-3 U/µg).
in space
on earth
U 3 1 0.3 0.1 0 L M L 0 0.1 0.3 1 3 U
multimeric ligated products
ocDNA
linear DNA
ccDNA
Fig. 3. Rejoining of DNA DSBs. Typical pattern
after gel electrophoresis. Lane U, untreated pRC
lane L, pRC digested by Sma I lane M, molecular
markers (?DNA-Hind III fragments). 0, 0.1, 0.3, 1
and 3, concentration of T4 DNA ligase (U/?g).
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Result 4
We found no effect of microgravity on rejoining
of DNA DSBs in space.
100
ligated products
75
50
Relative amounts ()
ccDNA
25
0
0.1
1
0.3
3
T4 DNA ligase (U/?g)
Fig. 4. Rejoining of DNA DSBs. Measurement of the
density of DNA bands produced by ligation at room
temperature (21.6-22.1C) for 10 h. Closed
symbols, on earth open symbols, in space.
Circles, ligated products (ccDNA, ocDNA and
multimeric ligated products) triangles, ccDNA.
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Future study
Space radiation includes heavy ions and other
types of high LET radiation during space flight.
It has been reported that restriction enzymes
produced DSBs which differed from those induced
by high-energy irradiation (Obe et al. 1992).
Therefore, the effect of space environment
consisting of microgravity and space radiation on
inappropriate rejoining frequency should be
studied in order to provide data useful for
determining how to maintain human health during
prolonged stays in space. Furthermore, in order
to check for microgravity effects in space, one
should perform experiments under microgravity and
compared them with experiments in the same space
environment with artificial 1g. One might still
question the relevance of the present simple in
vitro experiments in the light of the complexity
of the types of damage induced in space and the
response to microgravity of an integrated
cellular system. It is evident that in living
cells the repair of DSBs would involve more
complicated enzymatic processing due to the
different nature of DSBs induced by various types
of space radiation (protons, charged particles
etc.) and multiple enzymatic steps (e.g.
recombination) involved in their repair. In the
International Space Station, further experiments
might help to clarify the effects of microgravity
on the repair of DNA damage.
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Conclusion
No significant effect of microgravity on ligation
of damaged DNA was found during space flight.
Therefore, other mechanisms must account for the
synergism between radiation and microgravity, if
it exists.