OGG1 Flashcards
where is the humna ogg1 located and mouse ogg1
chomosome 3p25.3 and mouse - 6E
855 amino acid similarirty
DNA domans on ogg1
serveral domains conserved for more glycoslayse enzymes
typical domain is helix-hairpin-helix domain followed by a glycine/proline rich loop and a conserved aspartic acid (GPD motfi)
what are the active residuse in ogg1
Lys 249 and Asp 268 in the GPD motif, important for glycosylase/ap lyase activity
other DNA elements on Ogg1
mitochondiral targeting signal at N terminal end and a NLS at the carboxy terminal end
no TATA like sequence, ony GC rich binding site ==> suggesting OGG1 is a house keeping gene
Ogg1 human isoforms
at least 13 iofomrs have been identifdied
8 major ones have been studies
in general 2 groups
type 1 vs. t2
most adundant - type 1 alpha (NLS, major form int he nucles)nd type 2 alpha (major mito form)
ncluding colon, stomach, liver, duodenum, uterus, vagina, bladder, kidney and breast (only tissues measured)
how large is the Ogg1 protein
~ 47 kDA
mouse ogg1 gene/isoforms
1.7 kB
mouse alpha-ogg1 is similar to structure of alpha-human ogg1
only 1 isoform to date, repairs both nuclear and mito DNA
expressed in all tissues tested to date
Ogg1 substrate specificty
Ogg1 efficiently repairs the 8-oxoG lesion, as well as its open ring formed == FapyG
FapgyG is formed at equlavent or higher levels than 8-oxoG under phsy and oxidative stress conditions
OGG1 primarily repairs 8-oxoG
substrate effeicny is highest for 8-oxoG across from C
decreased activity of 8-oxoG across from T or G
but no activity for A opposite from 8-oxoG
Ogg1 DNA glycosylase vs. AP lyase reaction
occurs at different rate, suggesting a 2-stage mechanism
interaction with AP endonuclease (APE1) may stimulate the relsease of 8-oxoG removel of base
catalytic mechanism of OGG1
Lys249 attackes the glycosidic bond of 8-oxoG to release the resiude. Asp268 is not full clear –> may act as a proton donating residue reaulting in the protonation of epsalon group of Lys249 which may stabiilizr the developing negative change leaving on the 8-oxoG group. After Lys249 attacks the C-1’ of the deoxyribose sugar, resulting in the formation of a covelently linked enzyme-substrate intermediate. This intermeidate rearranges to a Schiff base –> which undergoes a lyase reaction to expel the 8-oxoG base via beta-elinination by the opening of the ribose ring within the Lys249.
Ogg1 expression in humans - 4
ogg1 RNA was detected “strong” expression in the thymus, testis, kidney, intestine, brain, cerebellum
ogg1 mRNA was detected in metaphase II oocyte, ans in the lower blastocyst
overall, ogg1 mRNA levels were measured decreased in somatic cells of humasn with age (newboarn to 91 years) –> suggesting the highest activty if ogg1 is during the fetal period
did not vary with the cell cycle, but expression was following a circadian rhythm (higher at 8 am compared to 8 pm) pattern wasn’t seen for APEX1 or XRCC1
ogg1 expression in mice -> tissue expression
animals 6 months of age
measured in kidney, muscle, liver, testis and heart –> higest in the testis
brain and hear did not have higher ogg1 acticites compared to the kidney, muslce, liver,
nuclear activity was x2 as high as mito activity in the brains of 6 month old mice
in general -> brain nucear activity was higher than mito in all tissues, in liver the mito activty was higher than the brain
ogg1 expression in the fetal period
several studies show that the higest ogg1 expression and ogg1 activity during the fetal period
ogg1 activity was 2 fold times higher in GD 17 mouse fetal brains and livers compared to adult brian and livers. fetal brains and livers had similar activity (wong et al 2008)
larsen et al 2006 -> analysis of devloping brains from the age of gd 9 until 10 weeks of age, showed that ogg1 protein levels and activity were the highest in gd19 and declined with age -> steeply withini the first 2 weeks
smilar pattern for rats, measures from E17 to post natal day 30, the highest levels were found at E17 and declined in a dose dependent manner
anotehr study showed the GD 16 whole rate fetuses exhibited 3 to 15 fold higher mRNA levels and 2 to 3 fold higher OGG1 incision activy compared to the adult liver (riis et al 2002)
another study (englander and ma 2006) -> ogg1 mRNA levels were ~ 2fold higher in the GD 12 whole fetal rat hears compared to the cerebra of 3 moth old mice, where levels had decreased to 50% by PND 21
ogg1 activity by brain region
in 6 month old mice, the cerebellum had the highest amounts of nuclear activity, followed by the brain stem, cortex or hippocampus and caudate and putamen with a max 10-fold variation
the mito ogg1 activity did not sig differ among the young brain regions, but followed the same trend
18 month vs. 6 months -> mito all the brain regions in the 18 month old mice (expcet ofr caudate nucleus) exhbited an age-depednet decrase in Ogg1 activity, whereas the nuclear activity the dramtic declins ocured in the cerebellum and brain stem
protein interaction with ogg1
APE1 and XRCC1 - ber pathway, increase ogg1 activtyi cut homeobox 1 and 2 transcriptionl activators of many DNA damage response genes, but stimulate the activity of OGG1 wihtouth the involement of their transcriptional function Stabilin 1 (STAB1) a gneomic organizer and a transciptional regulator, ineracts with ogg1 to stimulate its activity various class 1 histone deacetylases (HDACs 1-3) bu only weekly ineracts with class 2 hdacs --> decreasing the ogg1 activity
protein modification of ogg1 (6)
PARP1 - poly [adp-ribose] polymerase 1 - involved in DNA damge sensing protein in DNA repair, interacts with OGG1 to inhibit its activity, interaction is further enhanced on oxidative stress
ogg1 is acetylated in vivo by HATs like cyclic amp response element proteins/p300 complex (CBP/p300), acetylation at lys 338/341 sig increased OGG1 activity by reducing the affinity of OGG1 for its AP site - increasing the rate of BER
deacetylation by deacetylase sirtin 1 (SIRT1) can reduce the DNA repair activity of OGG1
phosporylated by serine.theronine kinases –> cyclin dependent kinase 4 (CDK4) –>increase in glycosylase activity and AP lyase activity but no change when phosphorylated by protein kinase c (PKC) and c-ABL
o-linked-beta-n-acetlyglycosamine by OGT –> inhibition of OGG1 actibity
reversble oxidaton by ROS of the thiol groups on OGG1 –> decreases the glycosylase activity of OGG1 –> but once oxidative stress conditions are resolved ogg1 activity is restored
ogg1 degradation
ubiquitylated-dependent proteasomal degradation and calpain1-dependent proteolysis
calcium-dependent proease calpain 1
Ogg1 polymorphisms
predominant polymorphism is Ser236Cys variant, C/G polymorphism
22%-59% for ser/cys
3-33% cys/cys – 50% recued catalytic effiecny
environmental factors associated with ogg1 activity
cadmium exposure -> decrease in ogg1 activity
steps of short batch repair
Ogg1 is recruited to the site, removes the base, APE1 generated a double strand break, pol b inserts a single nucleotide, and LIGIIIalpha/XRCC1 complex ligates, required atp
steps of long path repair
ogg1 is recruited, removes the 8-oxoG, APE1 generates a double strand break, pol delta and epsilon with PCNA scaffolding protein fill in 2-11 nucleotides, the old strand is degraded by FEN1 and ligated by LIGI, no atp needed
how is ogg1 activity measured
• OGG1 activity assay which involves using a synthetic duplex with an 8-oxoG (middle) and a terminal biotin, can measure the length of the fragments if 8-oxoG is removed double stranded break. Chemiluminescence for streptavidin on UV crosslinked gel
t. How was the knockout mouse generated?
• Targeted the helix-hairpin-helix structure which is essential for catalytic activity
u. What are the phenotypes of the OGG1 KO mouse?
• deficiencies in Myh and Ogg1 predispose 65.7% of mice to tumors, predominantly lung and ovarian tumors, and lymphomas.
