Nucleic
Acids Res. 2006 Mar 20;34(5):1620-32. Print
2006. Diabetes.
2006 Apr;55(4):1022-8.
Dimerization and opposite base-dependent catalytic
impairment of polymorphic S326C OGG1 glycosylase.
Hill
JW, Evans
MK.
Laboratory of Cellular and Molecular Biology, National
Institute on Aging, National Institutes of Health,
Human 8-oxoguanine-DNA glycosylase (OGG1) is the
major enzyme for repairing 8-oxoguanine (8-oxoG), a mutagenic guanine base
lesion produced by reactive oxygen species (ROS). A frequently occurring
OGG1 polymorphism in human populations results in the substitution of
serine 326 for cysteine (S326C). The 326 C/C
genotype is linked to numerous cancers, although the mechanism of
carcinogenesis associated with the variant is unclear. We performed
detailed enzymatic studies of polymorphic OGG1 and found functional defects
in the enzyme. S326C OGG1 excised 8-oxoG from duplex DNA and cleaved abasic sites at rates 2- to 6-fold lower than the
wild-type enzyme, depending upon the base opposite the lesion. Binding
experiments showed that the polymorphic OGG1 binds DNA damage with
significantly less affinity than the wild-type enzyme. Remarkably, gel
shift, chemical cross-linking and gel filtration experiments showed that
S326C both exists in solution and binds damaged DNA as a dimer. S326C OGG1 enzyme expressed in human cells was
also found to have reduced activity and a dimeric
conformation. The glycosylase activity of S326C
OGG1 was not significantly stimulated by the presence of AP-endonuclease. The altered substrate specificity, lack
of stimulation by AP-endonuclease 1 (APE1) and
anomalous DNA binding conformation of S326C OGG1 may contribute to its
linkage to cancer incidence.
PMID: 16549874 [PubMed - indexed for MEDLINE]
Protection of INS-1 cells from free fatty
acid-induced apoptosis by targeting hOGG1 to mitochondria.
Rachek
LI, Thornley
NP, Grishko
VI, LeDoux
SP, Wilson
GL.
Department of Cell Biology and Neuroscience, College of Medicine,
University of South Alabama, Mobile, AL 36688, USA.
lrachek@jaguar1.usouthal.edu
Chronic exposure to elevated levels of free fatty
acids (FFAs) impairs pancreatic beta-cell
function and contributes to the decline of insulin secretion in type 2
diabetes. Previously, we reported that FFAs
caused increased nitric oxide (NO) production, which damaged mitochondrial
DNA (mtDNA) and ultimately led to apoptosis in
INS-1 cells. To firmly establish the link between FFA-generated mtDNA damage and apoptosis, we stably transfected INS-1 cells with an expression vector
containing the gene for the DNA repair enzyme human 8-oxoguanine DNA glycosylase/apurinic lyase
(hOGG1) downstream of the mitochondrial targeting sequence (MTS) from
manganese superoxide dismutase.
Successful integration of MTS-OGG1 into the INS-1 cellular genome was
confirmed by Southern blot analysis. Western blots and enzyme activity
assays revealed that hOGG1 was targeted to mitochondria and the recombinant
enzyme was active. MTS-OGG1 cells showed a significant decrease in
FFA-induced mtDNA damage compared with
vector-only transfectants. Additionally, hOGG1 overexpression in mitochondria decreased FFA-induced
inhibition of ATP production and protected INS-1 cells from apoptosis.
These results indicate that mtDNA damage plays a
pivotal role in FFA-induced beta-cell dysfunction and apoptosis. Therefore,
targeting DNA repair enzymes into beta-cell mitochondria could be a potential
therapeutic strategy for preventing or delaying the onset of type 2
diabetes symptoms.
PMID: 16567524 [PubMed - in process]
Type 2 diabetes
