DNA Repair & Mutagenesis Research Highlights
Title: Structural Studies of Oxidatively Damaged DNA Duplexes and their Repair by DNA Glycosylases
Background and Advances
Implications & Public Health Impact
Center Contribution
Key Researchers
Publication(s)
Grant Support
Title: Posttranslational Modification of a Human Protein that Initiates Repair of Oxidized Bases in the Genome
Background and Advances
Implications & Public Health Impact
Center Contribution
Key Researchers
Publication(s)
Grant Support
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Title: Structural Studies of Oxidatively Damaged DNA
Duplexes and their Repair by DNA Glycosylases
Background and Advances: Oxidative DNA damage plays
significant roles in a number of disease processes, including
carcinogenesis and neurodegenerative diseases such as Alzheimer’s
disease. There is also strong evidence for the role of this type of DNA
damage in the aging process. Oxidized cytosine products are shown to be
the major precursors for the GC to AT transition mutations, the most
frequently observed point mutation in aerobic organisms. 5-hydroxy
Uracil is among the most mutagenic, stable product resulting from the
oxidation of cytosines. NMR spectroscopy and theoretical calculations
reveal that 5- hydroxy uracil can pair with all for DNA bases, but forms
the most stable pair with Guanine, and the least stable pair with
Cytosine. These studies also showed that the presence of 5-hydroxy
uracil does not significantly perturb the DNA structure. NEIL2, is a DNA
repair enzyme that recognizes the oxidative cytosine lesions in DNA
duplexes and repairs the damage. Molecular modeling, based on the
structure of an E.coli homolog, reveals two distinct domains in human
NEIL2. Results from proteolytic digestions confirmed the observations
made from molecular modeling. The 198-residue N-terminal domain of NEIL2
has been cloned and over-expressed in E.coli, and the structure is being
studied by multi-dimensional NMR spectroscopy.
Implications and Public Health Impact: Understanding the
mechanisms of oxidative damage and the DA repair process, as well as how
the DNA repair enzymes locate their targets, might allow us to modulate
DNA repair and the knowledge could lead to drug development to treat
drug- and/or radiation- resistant tumors.
Center Contribution: Through the Pilot Project program, the
NIEHS center provided funding for the research to study the structure
and stabilities of the DNA duplexes containing oxidative cytosine
lesions. DNA duplexes containing 5-hydroxy uracil modifications were
chemically synthesized by the Synthetic Organic Chemistry core (SOC).
Key Researchers:
David Gorenstein, DNA Repair & Mutagenesis Research Core, Department of
Biochemistry & Molecular Biology.
Tapas Hazra, DNA Repair & Mutagenesis Research Core, Department of
Biochemistry & Molecular Biology.
Sankar Mitra, DNA Repair & Mutagenesis Research Core, Department of
Biochemistry & Molecular Biology.
Publication(s):
Varatharasa Thiviyanathan, Anoma Somasunderam, David E. Volk and
David G. Gorenstein, (2005) “5-Hydroxy Uracil Can Form Stable Base
Pairs With all Four Bases in a DNA Duplex”, Chem. Commun., 3, 400-402.
David Volk, Varatharasa Thiviyanathan, Anoma Somasunderam, Tapas
Hazra, Sankar Mitra and David G. Gorenstein, “Ab initio base
pairing energies of Uracil and 5-hydroxy uracil with standard DNA bases
at the BSSE-optimized MP2 theory level” submitted to Organic and
Biomolecular chemistry, 2006.
Grant Support:
NIH/NCI1 R01 CA81063
Repair of Mutagenic 8-oxoguanine in Mammalian Genomes
NIH/NIAID R01 A127744
Combinatorial and Rational Design of Aptamers Targeting HIV
Title: Posttranslational Modification of a Human Protein
that Initiates Repair of Oxidized Bases in the Genome
Background and Advances: Reactive oxygen species (ROS) are
continuously generated both endogenously, as by-products of respiration,
and exogenously, due to a variety of stresses, disease-associated
inflammation and environmental toxicants. ROS have been implicated in a
wide range of pathophysiological states, in particular sporadic cancer,
arthritis and cardiovascular and neurological diseases. Genotoxicity of
ROS results from their ability to induce a large number of oxidized
bases as well as DNA strand breaks in the genome, most of which (except
for the DNA double-strand breaks) are repaired via the base excision
repair (BER) pathway. BER is initiated with excision of the damaged
bases by DNA glycosylases which appear to be limiting in the repair
process because the damaged bases. In particular 8-oxoguanine (8-oxoG),
a predominant ROS product in vivo, accumulates in the genome under
various pathological conditions. 8-oxoG and many other oxidized bases
have abnormal base-pairing potential and could thus generate mutations
in replicating cells and mutant proteins even in non-replicating cells
because 8-oxoG will be mistranscribed as if it were thymine. Thus,
repair of this and other oxidized bases is of paramount importance in
maintaining functional integrity of organisms.
8-oxoganine is repaired primarily by 8-oxoguanine-DNA glycosylase
(OGG1) which is conserved in mammals and other eukaryotes. The 8-oxoG
level in the genome is a sensitive barometer of cellular exposure to
oxidative stress, and it appears likely that cells have evolved a rapid
response to remove the excess, genomic 8-oxoG generated after transient
oxidative stress. This is warranted in order to quickly reestablish
homeostasis after the stress.
A recent study jointly published by several Center investigators and
their associates has shown the presence of an unexpected cellular
mechanism for the rapid response in increasing repair of 8-oxoG. In
short, after the cells confront oxidative stress, increased levels of
OGG1, which could have enhanced the rate of 8-oxoG repair, is not
observed. This lack of change in OGG1 production is likely because OGG1
is rather stable and does not turn over rapidly. Instead, the Center
investigators observed that oxidative stress sets off a signaling
process for activating a transcriptional co-activator named p300. P300
has an additional activity as an enzyme to insert acetyl group to
proteins containing specific acetyl acceptor sequences, including OGG1.
Acetylation of OGG1 increases its turnover leading to enhanced repair of
8-oxoG. Acetylation is reversed by a class of enzymes named histone
deacetylases (HDAC). The NIEHS Center Investigators then showed that
OGG1 binds to some HDACs which could restore the original, unmodified
enzyme by removing the acetyl group and reestablish homeostasis. Thus,
acetylation/deacetylation acts as a novel regulatory switch for rapid
response in repair when confronted with exogenous oxidative stress.
Implications and Public Health Impact: While the observation
of a previously unknown regulatory mechanism for repair of genomic
damage needed for maintaining cellular homeostasis in the face of
oxidative stress is fundamental in nature, this finding has several
implications in public health and therapy. For example, from the
perspective toxicogenomics, it is important to test for variability in
individual response to acetylation of OGG1 and its impact on base damage
repair in stressed cells. It should be noted that the basal level of
repair of 8-oxoG and other oxidized bases is not affected by acetylation,
but by the absolute level of OGG1 and potential variation in its
polymorphism-dependent glycosylase activity, which is being pursued in
the NIEHS Toxicogenomics Program as a separate topic.
Because acetyltransfer activity of p300 and deacetylase activity of
HDACs should have global impact in the cells, it would be important to
test (using genomic and proteomic approaches) how oxidative stress
affects other cellular functions. Activation of acetyltransferase
activity of p300 by oxidative stress was published earlier; however, the
studies of the NIEHS Center Investigators have highlighted the issue of
p300/HDAC dependent regulation of cellular functions including DNA
repair.
Center Contribution: The work was carried out primarily by
Drs. Kishor Bhakat and Sanath K. Mokkapati in the laboratory of Dr.
Sankar Mitra, Director of DNA Repair and Mutagenesis Research Core of
the NIEHS Center. Dr. Hazra, another Investigator of this Core, has
carried out extensive studies of human OGG1 in the past and provided
reagents, expertise and helped in experimental design in this
collaborative effort. Dr. Istvan Boldogh, Director of Cell Biology
Service Core and Investigator in Asthma Pathogenesis Research Core in
the NIEHS Center, collaborated in studies involving quantitation of
8-oxoG, and immunocytochemical studies via confocal microscopy.
The UTMB NIEHS Center in Environmental Toxicology played a unique
role in execution of this project because of complementary expertise of
the investigators. The Cell Biology Service Core was directly involved
in the project. The Biomolecular Resources Facility Core was responsible
for identification of acetylacceptor sites in OGG1 using MALD1-TOF and
direct sequencing. The Molecular Genomics Core was responsible for
generating many recombinant plasmids, and characterizing those by DNA
sequencing.
Key Researchers:
Sankar Mitra, DNA Repair and Mutagenesis Research Core, Department of
Biochemistry & Molecular Biology
Tapas K. Hazra, DNA Repair and Mutagenesis Research Core, Department
of Biochemistry & Molecular Biology
Istvan Boldogh, Cell Biology Service Core and Asthma Pathogenesis
Research Core, Department of Microbiology and Immunology
Publication(s):
Bhakat, K. K., Mokkapati, S. K., Boldogh, I., Hazra, T. K., and
Mitra, S., (2006) “Acetylation of human 8-oxoguanine-DNA
glycosylase by p300 and its role in 8-oxoguanine repair in vivo”,
Molecular and Cellular Biology, In Press.
Grant Support:
NIH/NCI 1 R01 CA81063
Repair of Mutagenic 8-oxoguanine in Mammalian Genomes
NIH/NIA 1 P01 AG021830
Oxidative Stress, Mitochondrial Dysfunction and Aging
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