Carcinogenesis vol.17 no.ll pp.25Ol-25O5, 1996
Nitric oxide inhibits DNA ligase activity: potential mechanisms for
NO-mediated DNA damage
Maria Graziewicz, David A.Wink1>2 and Francoise Laval
U347 INSERM, Rue du General Leclerc, 94276 Le Kremlin-Bicetre, France
and 'Radiation Biology Branch, Building 10 B3-B69, National Institutes of
Health/National Cancer Institute, Bethesda, MD 20893, USA
2
To whom correspondence should be addressed
Nitric oxide-induced modifications of DNA occur either by
directly altering DNA chemically through reactive nitrogen
oxide species (RNOS) or indirectly by inhibiting various
repair processes. DNA ligases are enzymes which rejoin
single-strand breaks and are critical for DNA integrity
during processes such as gene transcription and repair.
The eukaryotic and T4 DNA ligases are active in the
presence of ATP and act in two steps: the formation of
protein-AMP intermediates, then the ligation of DNA
breaks. When T4 DNA ligase was exposed to the NO
generator DEA/NO (Et2N[NO(NO)]Na), a concentrationand time-dependent inhibition of these two steps, adenylylation of the protein and ligation of the substrate, was
observed. This inhibition was abated by the presence of
cysteine, suggesting that RNOS, rather than NO, mediated
the inhibition of the ligase activity. As mammalian and T4
DNA ligases act by the same mechanism, the inhibition of
DNA ligase may explain the increase in single-strand breaks
reported for cells exposed to NO and provides a mechanism
to increase DNA lesions without direct chemical modification of DNA by NO or RNOS.
ine-DNA methyltransferase can be inhibited by the reaction
of NO, (equation (1)) with thiol residues to form a Snitrosothiol (10). Furthermore, proteins that contain zinc finger
motifs can be inhibited by the nitrosation of thiol groups
coordinated to zinc metal, which results in zinc ion release
and loss of protein structural integrity (11) critical for DNA
binding, this is the case for the Escherichia coli DNA repair
protein formamidopyrimidine-DNA glycosylase (12). However, not all DNA repair proteins are inhibited by the presence
of NO, e.g. uracil DNA glycosylase and endonucleases HI and
IV (12), suggesting that the presence of NO inhibits DNA
interacting proteins which have specific structure and critical
amino acids. DNA repair proteins are inhibited at NO concentrations 1000-10 000 times less than that required for deamination of DNA bases and may offer an important alternative
mechanism for DNA damage at lower NO exposures.
DNA ligases are required to rejoin strand interruptions
formed transiently during replication, repair and recombination
(reviewed in 13). T4 DNA ligase and mammalian DNA ligases
act by an identical mechanism, with the formation of covalent
ligase-AMP and DNA-AMP reaction intermediates, using
ATP as the source of the AMP group (14). Sequence analysis
of the active sites of the proteins has shown the presence of a
critical lysine residue (reviewed in 13). In this paper, we report
that the activity of T4 DNA ligase is inhibited by the presence
of NO. Therefore, inhibition of this protein could provide
insight into potential toxic, genotoxic and mutagenic mechanisms of DNA damage which do not directly relate to NO or
RNOS modifying DNA.
Introduction
Several studies suggest that NO is the causative agent in a
number of patho-physiological events (1-3). In deciphering
potential toxicological mechanisms involving NO, a number
of biological targets for NO and its derived reactive intermediates have been identified that could account for NO
deleterious effects. Because NO is a small diatomic molecular
radical, the chemistry of this molecule is a strong determinant
of its role in biology (4). NO itself can react directly with
some biomolecules that contain metals, such as the hemecontaining enzyme guanylate cyclase. Yet, under aerobic conditions, NO can react with oxygen to form reactive nitrogen
oxide intermediates (RNOS*) (equation (1)) which have been
shown to oxidize and nitrosate biological molecules (4).
4NO + O2 -» NO, [presumably N2O3 (4)]
(1)
It was shown that these intermediates can damage macromolecules such as DNA on exposure to very high concentrations of NO (5-9). Further investigation of the chemistry of
these intermediates revealed that RNOS could also inhibit
some DNA repair proteins, in particular those that contain
thiol-dependent motifs. We have reported that C^-methylguan•Abbreviations: RNOS, reactive nitrogen oxide intermediates; DEA/NO,
(C2H3)2N[N(O)NOrNa+; DTT, dithiothrcitol; BSA, bovine serum albumin;
CHO, Chinese hamster ovary.
O Oxford University Press
Materials and methods
Chemicals
T4 DNA ligase was from Boehringer Mannheim. (dT)| 6 and poly(dA) were
from Pharmacia. (C2H5)2N[N(O)NOrNa+ (DEA/NO) was supplied by Dr
J.Saavedra and was prepared as previously described (15). [a- PJATP (3000
Ci/mmol) was from Amersham. Dithiothrcitol (DTT) and bovine serum
albumin (BSA) were purchased from Sigma Chemical Co.
Incubation of DNA ligase with NO
DEA/NO was dissolved in a basic solution (pH 11.5) at a concentration of
25 mM. The T4 DNA ligase (1 jtl, 6 U/nl, 0.45 Jig protein) was diluted in a
final volume of 6 |il in a buffer containing 70 mM HEPES, pH 7.5, 10 mM
MgCl2 and increasing amounts of DEA/NO (final pH 7.7). After 15 min at
25°C, the protein was immediately used to measure either the formation of
DNA ligase-AMP intermediates or ligase activity.
Formation of DNA ligase—AMP intermediates
T4 DNA ligase (2 U, 0.150 |ig protein treated or not with DEA/NO) was
incubated at 25°C for 15 min in a buffer containing 66 mM HEPES, pH 8.0,
10 mM MgClj and [a-32P]ATP diluted with non-radioactive ATP to a sp. act
of 250 Ci/mmol and added to a final concentration of 2 nM ATP (16). The
final reaction volume was 10 (0.1. The reaction products were analyzed by
electrophoresis through SDS-polyacrylamide gels. The DNA ligase-AMP
complex was visualized by autoradiography and quantitated by densitometry.
DNA ligase activity determination
The substrate was [5'-32P]oligo(dT)i6poly(dA) prepared as described (10).
T4 DNA ligase (0.6 U) was incubated in a buffer containing 66 mM HEPES,
pH 7.5, 5 mM MgCl2 and 35 \iM ATP, the substrate (-40 000 c.p.m.), in a
final volume of 20 ill. After 15 min at 37°C, the reaction was stopped by
2501
M.Graziewicz, D.A.WInk and F.Laval
DEA/NO
(mM)
0
0.1
0.5
100
2.5
(A)
Z
D
Fig. 1. Inhibition of enzyme-adenylate formation by NO. T4 DNA ligase
was incubated or not with increasing DEA/NO concentrations, as described
in Materials and methods. After 15 min, [a-32P]ATP was added and the
incubation continued for 15 min. The enzyme-adenylylate complexes were
separated by electrophoresis and detected by autoradiography.
0.0
05
10
1.5
2.0
[DEA/NO] (mM)
25
100
adding 20 \l\ formamide dye and heating at 95°C for 10 min. The reaction
products were then separated through 15% polyacrylamide-8 M urea gels.
The radioactive multimers were detected by autoradiography and quantitated
by densitometry.
The DNA ligase activity was measured in Chinese hamster ovary (CHO)
cell extracts as described (17). Briefly, exponentially growing cells were
trypsinized, suspended in a buffer containing 66 mM Tris, pH 7.6, 50 mM
MgClj and 10% glycerol and disrupted by sonication. Cell debris was removed
by centrifugation. Cell extract was incubated or not with DEA/NO (2.5 mM)
for 15 min at 37°C, then with the radioactive oligo(dT)poly(dA) substrate
for different lengths of time. The reaction was stopped by heating at 68°C
for 30 min, then the samples were treated with 2.5 U alkaline phosphatase
for 1 h at 37°C. After addition of BSA and 10% tnchloroacetic acid, the acidprecipitable material was collected on filters and the radioactivity determined.
5
Results
Influence of NO on DNA ligase adenylylation
The reaction mechanism of ligase involves, in a first step, the
formation of a covalent ligase-AMP intermediate with a
lysine-adenylate phosphoamide bond (14). To measure the
influence of NO on this step of the reaction, T4 DNA ligase
was treated with various DEA/NO concentrations for 15 min
at 25°C prior to incubation with [a-32P]ATP. As shown in
Figure 1, formation of DNA ligase-adenylate was inhibited in
the presence of NO. The amount of adenylylated protein
decreased with DEA/NO concentration. The amount of DEA/
NO required to inhibit the activity by 50% (IC50) was 0.6 mM
DEA/NO (Figure 2A). To correlate the release of NO from
DEA/NO with this inhibition, a time-dependent experiment
was performed. Incubating the protein with DEA/NO (1 mM)
for increasing time intervals resulted in an increased inhibition
of adenylylation (Figure 2B), whereas a 5 min DEA/NO
treatment resulted in nearly 50% inhibition. The amount of
NO released from DEA/NO was calculated using the formula
(10,15) NOtWaJ = £NOCO(1 - e-*7), where k = 0.0005/s, Co =
1 mM and £ N O = 0.89. The calculated total amount of NO
released over 5 min was 130 ± 0.03 (imol/1. The amount of
NO released from 0.6 mM DEA/NO in 15 min was 190 ±
0.05 nmol/1. There was close correlation between the amounts
of NO at these IC^ values obtained in Figure 2A and B and
a correlation between the rate of NO release and inhibition of
the adenylylation reaction. The amount of NO required to
inhibit adenylylation was similar to that for inhibtion of the
alkyltransferase (90 |imol/l) (10) and Fpg protein (120 (imol/
1) activities (12).
Influence of NO on the DNA ligase activity
The enzyme was pretreated for 15 min at 25°C with various
concentrations of DEA/NO, then its enzymatic activity was
measured by the joining of single-strand interruptions in
2502
10
TIME
15
(mm)
Fig. 2. Influence of NO on the formation of T4 DNA ligase-AMP
intermediates Equal amounts of ligase (0.150 |ig protein) were treated or
not with increasing DEA/NO concentrations for 15 min then incubated with
[a-32P]ATP for 15 min at 25°C. After electrophoresis, the adenylylated
ligase was quantitated by densitometry (A) or the DNA ligase was treated
for increasing times with DEA/NO (1 mM) (B). Results are represented as a
percentage of the control value without DEA/NO exposure.
the double-stranded substrate [5'-32P]oligo(dT)i6poly(dA).
Separation of the multimers in denaturing polyacrylamide gels
showed that DEA/NO treatment decreased the activity of the
protein (Figure 3). There was a dose-dependent inhibition with
an ICso of -0.5 mM DEA/NO (Figure 4A). The total amount
of NO released under these conditions using the above formula
was 160 ± 30 |imol/l. When the protein was exposed to 1 mM
DEA/NO for various time intervals (Figure 4B), there was a
time-dependent decrease in its activity, with 50% inhibition
observed after ~7 min. The total amount of NO released under
these conditions, calculated from the above formula, was 170
± 30 (imol/1. Comparing the amount of NO released at the
IC50 values in the timing experiment (Figure 4B) versus the
dose experiment (Figure 4A), there was again a correlation
between the total amount of NO released and inhibition of the
enzyme activity. When the DEA/NO solution was allowed to
stand at 50°C at pH 7.7, which results in DEA/NO decomposition (15), no inhibition of ligase activity was observed (Table
I), showing that the inhibition process is actually due to NO
release. Comparison of the NO required for adenylylation and
ligase inhibtion was nearly identical, suggesting that a common
pathway of inhibition by NO occurs for both processes.
It has been shown that NO inhibits various enzymes, not
by a direct chemical reaction with the protein, but through the
formation of RNOS (4). To determine whether NO or RNOS
derived from the NO/O2 reaction was responsible for DNA
Nitric oxide inhibits DNA Ugase activity
E 6000 •
4000 •
2000
30
10
20
Incubation Time (min)
Fig. 3. Inhibition of the DNA ligase activity by DEA/NO. Equal amounts of
DNA ligase were treated or not with incresasing DEA/NO concentrations,
then incubated with the substrate [5'-32P]oligo(dT)16-poly(dA) for 15 min at
37°C. The oligo<dT)j6 multimers were separated in polyacrylamide-urea
gels and visualized by autoradiography. Lane 1, control without enzyme;
lane 2, T4 DNA ligase without DEA/NO; lanes 3-5, incubated with 0.5
(lane 3), 1 (lane 4) or 1.5 mM (lane 5) DEA/NO.
Fig. 5. Influence of NO on DNA ligase activity from cell extracts. DNA
ligase activity in CHO cell extracts preincubated for 15 min at 37°C without
(filled triangles) or with 2.5 mM DEA/NO (open triangles). The activity
was measured by incubating the extract (10 ug proteins) with [5'32
P]oligo(dT)16-poly(dA). For details see Materials and methods.
Table L Influence of DEA/NO on T4 DNA ligase activity in the presence
of cysteine or BSA.
DNA ligase activity"
Control
Protein
Protein
Protein
Protein
protein
+ DEA/NO (1 mM)
+ expired DEA/NO (1 mM)b
+ DEA/NO (1 mM) + cysteine (10 mM)
+ DEA/NO (1 mM) + BSA (1 mg/ml)
6.5 (100)
0.58 (9)
5.9 (90)
4.4 (68)
1.17(18)
1
fmol oligo(dT)poly(dA) ligated in 15 min at 37°C by 0.1 U T4 DNA
ligase.
b
DEA/NO was incubated at 50°C at pH 7.7 for 45 min prior to addition of
the protein.
10
TIME
20
(min)
Fig. 4. Influence of NO on T4 DNA hgase activity. The protein was
incubated in the presence of increasing amounts of DEA/NO for 15 min
before measuring its activity. The oligo(dT)|6 multimers were separated by
electrophoresis and quantitated by densitomctry (A) or the protein was
incubated with DEA/NO (1 mM) for various lengths of time, then with the
substrate for 15 min at 37°C (B).
ligase activity inhibition, the protein was incubated with DEA/
NO in the presence of cysteine. We have previously shown
that cysteine has a high affinity for RNOS species while not
directly reacting with NO (18). When the T4 DNA ligase was
incubated with 10 mM DEA/NO in the presence of 10 mM
cysteine, significant protection of the activity was observed
(Table I). A similar protection in the presence of cysteine was
observed for the adenylylation reaction (data not shown).
This result suggests that RNOS could be responsible for the
inactivation of the protein, possibly resulting in a 5-nitrosothiol
protein adduct. These 5-nitrosothiols decompose with time,
which can result in restoration of activity in the presence of
DTT (10). However, exposing the enzyme to 1 mM DEA/NO
and then measuring its activity at various time intervals for
24 h revealed no recovery of the activity, even in the presence
of DTT (data not shown). The effect of DEA/NO was also
measured in the presence of BSA. Addition of BSA (1 mg/
ml) in the DEA/NO-containing buffer showed no effect on
either formation of protein-AMP intermediates (data not
shown) or on ligase activity (Table I). To confirm that the
presence of a large excess of protein is not sufficient to protect
the enzyme against inactivation by NO, extracts from CHO
cells were incubated with DEA/NO for 15 min at 37°C; ligase
activity was found to be decreased by ~65%, showing that
NO is able to reduce the activity even in the presence of high
concentrations of proteins.
Discussion
The presence of NO has been shown to inhibit specific enzymes.
Although DNA repair enzymes such as endonucleases HI
and IV or uracil DNA glycosylase are not inhibited by NO
(12), proteins which contain critical thiol residues are inhibited.
2503
M-Graziewicz, D.A.Wink and F.Laval
(4)
various biological conditions. It has been shown that activated
neutrophils and macrophages generate signifigant nitrosation
of amines (23,24), while RNOSs such as peroxynitrite have
been shown to be formed in alveolar macrophages (25). It has
been suggested that nitrosation of morpholine from lipopolysaccharide-stimulated RAW macrophages occurs via equations
(1) and (2) as well as equations (3)-(6) (26). In addition to
cell experiments, nitrosation of amines and thiols has been
shown to occur in chronically infected tissue (27-29): It would
appear that amine nitrosation occurs in a variety of biological
conditions which may result in inactivated ligase function.
(The rate of the NO formation from stimulated macrophages
and neutrophils is -0.5—1 nmol/min/106 cells. Calculating this
value in terms of the cell volume there is ~0.5-l mmol/1/min
NO produced. In these experiments, 0.02-0.4 mmol/1/min were
produced during the NO exposure, suggesting that there is
sufficient NO generated from these stimulated immune cells
to inhibit ligase.)
Different DNA ligases have been found in mammalian cells
(30). DNA ligase I is involved in the ligation of replication
intermediates into high molecular weight DNA during DNA
replication (31). A role in meiotic recombination has been
suggested for DNA ligase II (32) and DNA ligase ITI activity
has been related to DNA repair (33,34). Recently, a fourth
DNA ligase has been identified (35). It has also been shown
that an inherited molecular defect in DNA ligase I resulted in
immunosuppression, lymphoma and hypersensitivity to DNA
damaging agents (36). Therefore, inhibition of DNA ligase
activity could play a crucial role in the cell and could explain
some previous observations. Exposure of cells to NO results
in an increased number of DNA single-strand breaks (6).
However, when purified DNA is exposed to NO, even at doses
resulting in an RNOS concentration of 1 M, there is no
formation of single-strand breaks (9). This implies that direct
chemical modification of DNA by NO or RNOS produces
DNA breaks in vitro. Our results suggest that NO inhibits
DNA ligase activity resulting in the accumulation of DNA
breaks formed either during transcription or repair, thus
explaining these lesion observed in cells. Another implication
of NO-mediated ligase inhibition is that genotoxicity resulting
from either RNOS or reactive oxygen species might be
amplified. It has been shown that activated macrophages result
in deamination of DNA (37). Furthermore, under conditions
where superoxide and peroxide are formed, the presence of
NO can induce single- and double-strand breaks, possibly by
peroxynitrite or mobilization iron, which furthers Fenton-type
oxidation in the nucleus (37-40). Repair of both abasic
formation via deamination and direct oxidation of deoxyribose
involve ligase. Thus, inhibition of ligase may increase the
number of these lesions. By whichever mechanism, the increase
in DNA breaks due to NO-mediated inhibition of ligase could
in turn activate the tumor supressor gene p53 (41) or activate
poly(ADP-ribose) synthesis (42).
(5)
Acknowledgements
This is the case for the C^-methylguanine-DNA methyltransferase (alkyltransferase), whose active site contains a cysteine
residue (10). This inhibition is expected because the RNOS
formed in the NO/O2 reaction has a high affinity for thiolcontaining residues (18). The resulting product forms a Snitrosothiol which either inactivates a thiol or can labilize zinc
from the zinc finger motifs (11,12).
Our results show that T4 DNA ligase activity was inhibited
in the presence of NO. Recovery of the activity was observed
neither with time nor in the presence of DTT, suggesting that
the reaction is different from that observed in the case of the
alkyltransferase protein. A critical lysine residue is present in
the active site of T4 DNA ligase protein (13). This residue
initially forms an intermediate with an adenyl group from
ATP, then the adenyl group is transferred to the 5'-P end of
DNA. In the final step, unadenylylated ligase is required for
generation of a phosphodiester bond. The initial adenylylation
of the lysine is therefore a crucial step. In the presence of NO,
nitrosation of the lysine would form a primary nitrosamine,
which would rapidly rearrange to the diazonium salt, followed
by hydrolysis to yield the corresponding hydroxy adduct
(equation (2)).
Protein-NH2 + NOX -» protein-NH-NO -> protein-OH + N2
(2)
It has been reported that the lysine residue is partially in the
deprotonated form in the ligase protein (14). The lone pair of
electrons of the nitrogen atom that bind to the adenylate group
via nucleophilic attack provides a site for electrophilic attack.
In the presence of NO, nitrosation of the partially deprotonated
amine could occur via NOX, which then results in a primary
nitrosamine. Since primary nitrosamines undergo deamination
(19), this nitrosamine would result in a hydroxy group (equation
(2)). This chemistry represents a new mechanism by which
NO (via RNOS) can interact with proteins, suggesting that the
pH of the protein site of the critical lysine residue is an
important determinant for interaction with RNOS.
Nitrosation of biological substances can arise through several
different chemical mechanims in vivo. In this paper, the
reaction between NO and oxygen to form isomers of N2O3 (4)
was used to assess the chemistry of nitrosation on ligase
(equation (1)). Another potential in vivo source of nitrosating
species is derived from acidic nitrite, which might be expected
in areas of the gastrointestinal tract or in a phagocytic macrophages. Alternatively, N2O3 can be formed from the interaction
of NO and superoxide, which results in peroxynitrite (OONO~).
NO + O 2 " -> OONO-
(3)
In the presence of excess NO or superoxide, peroxynitrite is
converted to nitrogen dioxide (20-22).
H+
OONO- + NO -> NO 2 + NO2"
H+
OONO- + O2"-> NO 2
Nitrogen dioxide can rapidly react with NO to form the
nitrosating species, N2O3 (equation (6)).
NO2 + NO -> N2O3
(6)
The presence of N2O3 could then nitrosate the lysine residue
of ligase, thus inactivating the enzyme.
Nitrosation of amines and thiols have been shown under
2504
This work was supported by grants from INSERM, the ARC (Villejuif) and
the EC. MG was supported by a fellowship from the EC.
References
1 .Moncada,S., Palmer.R.M J. and Higgs.E.A. (1991) Nitric oxide: physiology,
pathophysiology, and pharmacology. Pharmacol Rev., 43, 109-142.
2. Feldman.P.L., Gnffith.O.W. and Stuehr,DJ. (1992) The surprising life of
nitric oxide. Chem. Eng. News, 20 December, 26-38.
Nitric oxide inhibits DNA ligase activity
3.Ignarro,LJ. (1990) Biosynthesis and metabolism of endothelium-derived
nitric oxide. Annu. Rev. Pharmacol. Toxicol., 30, 535-560.
4. Wink,D.A., Hanbauer.I., Grisham.M.B. et al. (1996) The chemical biology
of NO. Insights into regulation, protective and toxic mechanisms of nitric
oxide. Curr. Topics Cell. Regulat., 34, 159-187.
5.Wink,D.A., Kasprzak,K.S., Maragos.C.M. et al. (1991) DNA deaminating
ability and genotoxicity of nitric oxide and its progenitors. Science, 254,
1001-1003.
6.Nguyen,T., Brunson,D., Crespi.C.L., Penman.B.W., WishnokJ.S. and
Tannenbaum,S.R. (1992) DNA damage and mutation in human cells
exposed to nitric oxide. Proc. Natl Acad. Sci. USA, 89, 3030-3034.
7.Arroyo,P.L., Hatch-Pigott.V., Mower,H.F. and Cooney.R.V. (1992)
Mutagenicity of nitric oxide and its inhibition by antioxidants. Mutai.
Res., 281, 193-202.
8.Routledge,M.N., Wink,D.A., Keefer.L K. and DippleA (1994) DNA
sequence changes induced by two nitric oxide donor drugs in the supF
assay. Chem. Res. Toxicol, 7, 628-632.
9.Routledge,M.N., WinkJJ.A., Keefer.L.K. and Dipple.A. (1993) Mutations
induced by saturated aqueous nitric oxjde in the pSP189 supF gene in
human Ad293 and E.coli MBM7070 cells. Carcinogenesis, 14, 1251-1254.
10. Laval.F. and Wink.D.A. (1994) Inhibition by nitric oxide of the repair
protein O6-methylguamne-DNA-methyltransferase. Carcinogenesis, 15,
443-447.
ll.Kroncke,K.-D.,
Fechsel,K.,
Schmidt,T.,
Zenke.F.T.,
Dasting.I.,
WesenerJ.R., Bettermann.H., Breunig.K.D. and Kolb-Bachofen,V. (1994)
Nitric oxide destoys zinc-finger clusters inducing zinc release from
metallothionein and inhibition of the zinc finger-type yeast transcription
activator LAC9. Biochem. Biophys. Res. Commun., 200, 1105-1110.
12.Wink,D.A. and LavalJ. (1994) The Fpg protein, a DNA repair enzyme,
is inhibited by the biomediator nitric oxide in vitro and in vivo.
Carcinogenesis, 15, 2125-2129.
13.Lindahl,T. and Bames,D.E. (1992) Mammalian DNA ligases. Annu. Rev.
Biochem., 61, 251-281.
14.Engler,MJ. and Richardson,C.C. (1982) DNA ligase. In Boyer.P.D. (ed.),
The Enzymes. Academic Press, New York, NY, pp 3-29.
15. Maragos.C.M., Morley,D., Wink,D.A. et al. (1991) Complexes of NO with
nucleophiles as agents for the controlled biological release of nitric oxide.
Vasorelaxant effects. J. Med. Chem., 34, 3242-3247.
16.Prigent,C, Satoh.M.S., Daly.G., Bames.D.E. and LindahlJ. (1994)
Aberrant DNA repair and DNA replication due to an inherited enzymatic
defect in human DNA ligase I. Mol. Cell. Bioi, 14, 310-317.
17.Petrini,Y.H., Huwiler.K.G. and Weaver.D.T (1991) A wild-type DNA
ligase I gene is expressed in Bloom's syndrome cells. Proc. Natl Acad.
Sci. USA, 88, 7615-7619.
18.Wink,D.A., Nims,R.W., DarbyshireJ.F. et al. (1994) Reaction kinetics for
nitrosation of cysteine and glutathione in aerobic nitric oxide solutions at
neutral pH. Insights into the fate and physiological effects of intermediates
generated in the NO/O2 reaction. Chem. Res. Toxicol., 7, 519-525.
19.Williams,D.L.H. (1988) (ed.), Nitrosation. Cambridge University Press,
Cambridge, UK.
20. BeckmanJ.S., ChenJ., Ischiropoulos.H. and CrowJ.P. (1994) Methods
Enzymol., 233, 229-240.
21.Koppenol,W.H.,
Moreno JJ.,
Pryor.W.A., Ischiropoulus.H.
and
BeckmanJ.S. (1992) Peroxynitrite, a cloaked oxidant formed by nitric
oxide and superoxide. Chem. Res. Toxicol., 5, 834-842.
22.Miles,A.M., Bohle.D.S., Glassbrenner,P.A., Hansert,B., Wink.D.A. and
Gnsham.M.B. (19%) Modulation of superoxide-dependent oxidation and
hydroxylation reactions by nitric oxide. J. Biol. Chem., 271, 40-47.
23.Miles,A.M., Gibson.M., Krishna,M., CookJ.C, Pacelli.R., Wink,D.A. and
Grisham.M.B. (1995) Effects of superoxide on nitric oxide-dependent Nnitrosation reactions. Free Radical Res., 233, 379-390.
24.Marletta,M.A. (1988) Mammalian synthesis of nitrite, nitrate, nitric oxide
and W-nitrosating agents. Chem. Res. Toxicol., 1, 249—257.
25. Ischiropoulos.H., Zhu.L. and BeckmanJ.S. (1992) Peroxynitrite formation
from macrophage-derived nitric oxide. Arch. Biochem. Biophys., 298,
446-451.
26. Lewis.R.S., Tamir,S., Tannenbaum.S.R. and Deen.W.H. (1995) Kinetic
analysis of the fate of nitric oxide synthesized by macrophages in vitro.
J. Biol. Chem., 270, 29350-29355.
27.Liu,R.H., Baldwin.B., Tennant,B.C. and HotchkissJ.H. (1991) Elevated
formation of nitrate and /V-nitrosodimethylamine in woodchucks (Marmota
monax) associated with chronic woodchuck hepatitis virus infection.
Cancer Res., 51, 3925-3929.
28.Liu,R.H., JacobJ.R., Tennam,B.D. and HotchkissJ.H. (1992) Nitrite and
nitrosamine synthesis by hepatocytes isloated from normal woodchucks
(Marmota monax) and woodchucks chronically infected with woochuck
hepatitis virus. Cancer Res., 52, 4139-4143.
29.Gaston,B., ReillyJ., DrazenJ.M. et al. (1993) Endogenous nitrogen oxides
and bronchodilator S-nitosothiols in human airways. Proc. Natl Acad. Sci.
USA.90, 10957-10961.
30.Tomkinson,A.E., Roberts.E., Daly.G., Tofty,N.F. and LindahlJ. (1991)
Three distinct DNA ligases in mammalian cells. J. Biol. Chem., 266,
21728-21735.
31.Li,C., GoodchildJ. and Banl,E.F. (1994) DNA ligase I is associated with
the 21S complex of enzymes for DNA synthesis in HeLa cells. Nucleic
Acids Res., 22, 632-638.
32.Higashitani,A, Tabata,S., Endo.H. and Hotta,Y. (1990) Purification of
DNA ligases from mouse testis and their behaviour during meiosis. Cell
Structure Fund., IS, 67-72.
33.Caldecott,K.W., McKeown.C.K., TuckerJ.D., Ljungquist,S. and
Thompson,L.H. (1994) An interaction between the mammalian DNA
repair protein XRCC1 and DNA ligase III. Mol. Cell. Biol., 14, 68-76.
34.Ljungquist,S., Kenne.K., 01sso,L. and Sandstrom,M. (1994) Altered DNA
ligase III activity in the CHO EM9 mutant. Mutat. Res., 314, 177-186.
35.Wei,Y., Robins,R, Carter,K. et al. (1995) Molecular cloning and expression
of human cDNAs encoding a novel DNA ligase IV and DNA ligase Hi,
an enzyme active in DNA repair and recombination. MoL Cell. Biol., 15,
345-367.
36.Bames,D.E., Tomkmson,A.E., Lehman.A.R., Webster, A.D.B. and
Lindahl.T. (1992) Mutations in the DNA ligase I gene of an individual
with immunodeficiencies and cellular hypersensitivity to DNA damaging
agents. Cell, 69, 495-503.
37.de Rojas-Walker.T., Tamir.S., Ji,H., WishnokJ.S. and Tannenbaum.S.R.
(1995) Nitnc oxide induces oxidative damage in addition to deamination
in macrophage DNA. Chem. Res. Toxicol., 8, 473-477.
38. King.P.A., Anderson.V.E., EdwardsJ.O., Gustafson.G., Plumb,R.C. and
SuggsJ.W. (1992) A stable solid that generates hydroxyl radical upon
dissolution in aqueous solution: reactions with proteins and nucleic acids.
J. Am. Chem. Soc, 114, 5430.
39.Salgo,M.G., Bermudez.E., Squadnto.G.L. and Pryor,W.A. (1995) Peroxynitrite causes DNA damage and oxidation of thiols in rat thymocytes.
Arch. Biochem. Biophys., 322, 500-505.
40. Salgo.M.G., Stone.K., Squadrito.G.L., BattistaJ.R. and Pryor.W.A. (1995)
Peroxynitrite causes DNA nicks in plasmid pBR322. Biochem. Biophys.
Res. Commun., 210, 1025-1030.
41.Messmer,U.K., Ankarcrorwjvl., Nicotera,P. and Brune.B. (1994) p53
expression in nitric oxide-induced apoptosis. FEBS Lett., 355, 23-26.
42.ZhangJ., Dawson.V.L., Dawson.T.M. and Synder.S.H. (1994) Nitric oxide
activation of poly(ADP-ribose) synthetase in neurotoxicity. Science, 263,
687-689.
Received on May 14, 1996; revised on July 4, 1996; accepted on July 3, 1996
2505