3 Biomarkers of Oxidative Damage in Human Disease_dalle Done
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Transcript of Oxidative Damage of DNA Oxidative damage results from aerobic metabolism, environmental toxins,...
Oxidative Damage of DNA
Oxidative damage results from aerobic metabolism, environmental toxins, activated macrophages, and signaling molecules (NO)
Compartmentation limits oxidative DNA damage
guanine 8-oxoguanine
The most common mutagenic base lesion is 8-oxoguanine
from Banerjee et al., Nature 434, 612 (2005)
Oxidation of Guanine Forms 8-Oxoguanine
Repair of 8-oxo-G
8-oxoguanine DNA glycosylase/-lyase (OGG1) removes 8-oxo-G and creates an AP site
Replication of the 8-oxoG strand preferentially mispairs with A and mimics a normal base pair and results in a G-to-T transversion
MUTYH removes the A opposite 8-oxoG
from David et al., Nature 447, 941 (2007)
Free dNTPs are much more susceptible to oxidative damage than bases in duplex DNA
Oxidized precursors are misincorporated and are mutagenic
MTH1 removes oxidized nucleotides from the pool
from Dominissini and He, Nature 508, 191 (2014)
MTH1 Prevents Incorporation of Oxidized dNTPs into DNA
MTH1 is not essential in normal cells
Higher levels of ROS in cancer cells causes a non-oncogene addiction to MTH1
from Gad et al., Nature 508, 215 (2014)
Inhibition of MTH1 Selectively Kills Cancer Cells
UV-Irradiation Causes Formation of Thymine Dimers
from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-38
Nonenzymatic Methylation of DNA
Formation of 600 3-me-A residues/cell/day are caused by S-adenosylmethionine
3-me-A is cytotoxic and is repaired by 3-me-A-DNA glycosylase
7-me-G is the main aberrant base present in DNA and is repaired by nonenzymatic cleavage of the glycosyl bond
Effect of Chemical Mutagens
Nitrous acid causes deamination of C to U and A to HX
U base pairs with AHX base pairs with C
Repair Pathways for Altered DNA Bases
from Lindahl and Wood, Science 286, 1897 (1999)
Direct Repair of DNA
Photoreactivation of pyrimidine dimers by photolyase restores the original DNA structure
O6-methylguanine is repaired by removal of methyl group by MGMT
1-methyladenine and 3-methylcytosine are repaired by oxidative demethylation
Base Excision Repair of a G-T Mismatch
At least 8 DNA glcosylases are present in mammalian cells
DNA glycosylases remove mismatched or abnormal bases
AP endonuclease cleaves 5’ to AP site
AP lyase cleaves 3’ to AP site
from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-36
BER works primarily on modifications caused by endogenous agents
Each glycosylase has limited substrate specificity, but there is redundancy in damage recognition
DNA Glycosylases
from Xu et al., Mech.Ageing Dev. 129, 366 (2008)
Mechanism of hOGG1 Action
hOGG1 binds nonspecifically to DNA
Contacts with C results in the extrusion of corresponding base in the opposite strand
G is extruded into the G-specific pocket, but is denied access to the oxoG pocket
oxoG moves out of the G-specific pocket, enters the oxoG-specific pocket, and excised from the DNA
from David, Nature 434, 569 (2005)
UV-induced pyrimidine dimers
Nucleotide Excision Repair
Bulky adducts
Repairs helix-distorting lesions
Intrastrand crosslinks
ROS-generated cyclopurines
Global Genome NER – Damage Recognition
Probes for helix distorting lesions
XPC is the damage sensor which finds the ssDNA gap caused by disrupted pairing
UV-DDB (DDB1 and DDB2) can stimulate XPC binding by extruding the lesion to create ssDNA
from Marteijn et al., Nature Rev.Mol.Cell Biol. 15, 465 (2014)
Transcription-coupled NER – Damage Recognition
Repairs transcription-blocking lesions
CSB, UVSSA and USP7 interact with Pol II
With CSA, promotes backtracking of Pol II to expose lesion
from Marteijn et al., Nature Rev.Mol.Cell Biol. 15, 465 (2014)
TFIIH complex is recruited to the lesion
XPB and XPD are helicases with opposite polarity
XPD verifies the existence of lesions and XPA binds to altered nucleotides
XPG nuclease binds to the complex
RPA protects the undamaged strand from nucleases
NER – Lesion Verification
from Marteijn et al., Nature Rev.Mol.Cell Biol. 15, 465 (2014)
NER – Strand Excision
XPF nuclease is recruited by XPA and directed to the damaged strand by RPA
XPF and XPG excises the lesion
from Marteijn et al., Nature Rev.Mol.Cell Biol. 15, 465 (2014)
PCNA recruits DNA polymerase to fill ss gap
Nick is sealed by DNA ligase
NER – Gap Filling and Ligation
from Marteijn et al., Nature Rev.Mol.Cell Biol. 15, 465 (2014)
NER is stimulated by an open chromatin environment
UV-DDB ubiquitylates core histones and associates with PARP1 which PARylates chromatin
Histone acetylation stimulates NER
Chromatin remodelling complexes displace nucleosomes
Chromatin Dynamics in GG-NER
from Marteijn et al., Nature Rev.Mol.Cell Biol. 15, 465 (2014)
Clinical Implications of Defective NER
GG-NER is elevated in germ cells to maintain the entire genome to prevent mutagenesis
TC-NER is elevated in somatic cells to repair expressed genes to prevent cell death
Defective GG-NER increases cancer predisposition
Defective TC-NER causes premature cell death, neurodegeration and accelerates aging
Xeroderma pigmentosum
Cockayne Syndrome
Mismatch Repair
Repairs DNA replication errors and insertion-deletion loops
Decreases mutation frequency by 102 - 103
Plays a role in triplet repeat expansion, somatic hypermutation and class switch recombination
GATC sequences are methylated by dam methylase
Newly replicated DNA is transiently hemimethylated
MutS recognizes a mismatch or small IDL
MutS bends DNA, recruits MutL and forms a small dsDNA loop
MutH nicks the unmethylated GATC
Helicase unwinds the nicked DNA which is degraded past the mismatch
Gap is repaired by Pol III and ligase
from Marra and Schar, Biochem.J. 338, 1 (1998)
Mismatch repair in E. coli
Mismatch Repair in Eukaryotes
from Hsieh and Yamane, Mech.Ageing Dev. 129, 391 (2008)
MutS homologs recognize mismatch and form a ternary complex with MulL homologs and the mismatch
PMS2 is a mismatch-activated strand-specific nuclease, and the break is directed to the strand contain the preexisting nick
EXO1 excises the mismatch
The gap is filled in by PCNA, Poland DNA ligase
Defective mismatch repair is the primary cause of certain types of human cancers
Causes of and Responses to ds Breaks
Repair of DSBs is by homologous recombination or nonhomologous end joining
DSBs result from exogenous insults or normal cellular processes
DSBs result in cell cycle arrest, cell death, or repair
from van Gent et al., Nature Rev.Genet. 2, 196 (2001)
Initiation of Double-stranded Break Repair
from van Attikum and Gasser, Trends Cell Biol. 19, 204 (2009)
MRN complex recognizes DSB ends and recruits ATM
ATM phosphorylates H2A.X and recruits MDC1 to spread H2A.X
TIP60 and UBC13 modify H2A.X
MDC1 recruits RNF8 which ubiquitylates H2A.X
RNF168 forms ubiquitin conjugates and recruits BRCA1
ATM Mediates the Cell’s Response to DSBs
from van Gent et al., Nature Rev.Genet. 2, 196 (2001)
DSBs activate ATM
ATM phosphorylation of p53, NBS1 and H2A.X influence cell cycle progression and DNA repatr
ssDNAs with 3’ends are formed and coated with Rad51, the RecA homolog
Rad51-coated ssDNA invades the homologous dsDNA in the sister chromatid
The 3’-end is elongated by DNA polymerase, and base pairs with ss 3-end of the other broken DNA
DNA polymerase and DNA ligase fills in gaps
from Lodish et al., Molecular Cell Biology, 5th ed. Fig 23-31
Repair of ds Breaks by Homologous Recombination
Role of BRCA2 in Double-stranded Break Repair
BRCA2 mediates binding of RAD51 to ssDNA
RAD51-ssDNA filaments mediate invasion of ssDNA to homologous dsDNA
from Zou, Nature 467, 667 (2010)
from van Gent et al., Nature Rev.Genet. 2, 196 (2001)
Repair of ds Breaks by Nonhomologous End Joining
KU heterodimer recognizes DSBs and recruits DNA-PK
Mre11 complex tethers ends together and processes DNA ends
DNA ligase IV and XRCC4 ligates DNA ends
Translesion DNA Synthesis
from Sale et al., Nature Rev.Mol.Cell Biol. 13, 141 (2012)
Replicative polymerase encounters DNA damage on template strand
Replicative polymerase is replaced by TLS polymerase which inserts a base opposite lesion
Base pairing is restored beyond the lesion and replicative polymerase replaces TLS polymerase
TLS can occur in S or G2
Catalytic site of replicative polymerases is intolerant of misalignment between template and incoming nucleotide
TLS polymerases are recruited by interactions with the sliding clamp
There are multiple TLS polymerases
TLS polymerases have low processivity and low fidelity, and lack 3’-5’ exonucleases
TLS polymerases are selective for certain lesions
Most mutations caused by DNA lesions are caused by TLS polymerases
from Sale et al., Nature Rev.Mol.Cell Biol. 13, 141 (2012)
There are Multiple TLS Polymerases
TLS Polymerases Can Be Accurate or Error-prone
Pol bypasses an abasic site and often causes a -1 frameshift
Pol bypasses a thymine dimer and inserts AA
Pol is accurate with dA template and error-prone with dT template
Replicative polymerases insert dC or dA opposite 8-oxo-G, Pol inserts dC
The likelihood that TLS polymerases are error-prone depends on the nature of the lesion and the TLS polymerase that is utilized
Somatic Hypermutation of Ig Genes Depends on TLS Polymerases
from Sale et al., Nature Rev.Mol.Cell Biol. 13, 141 (2012)
Uracil DNA glycosylase forms an abasic site, and REV1 incorporates dC opposite the site
AID deaminates dC to dU
MMR proteins lead to the formation of a ss gap, PCNA is ubiquitylated, and Pol is recruited, generating mutations at A-T