oxidative damage of dna
DESCRIPTION
Oxidative Damage of DNA. Oxidative damage results from aerobic metabolism, environmental toxins, activated macrophages, and signaling molecules (NO). Compartmentation limits oxidative DNA damage. Oxidation of Guanine Forms 8-Oxoguanine. The most common mutagenic base lesion is 8-oxoguanine. - PowerPoint PPT PresentationTRANSCRIPT
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
Oxidation of dNTPs are Mutagenic
cGTP is oxidized to 8-OH-dGTP and is misincorporated opposite A
MutT converts 8-OH-dGTP to 8-OH-dGMP
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-adnosylmethionine
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)
Nucleotide excision repair mainly works on helix distortion and damage caused by environmental mutagens
Nucleotide Excision Repair
Recognition of Helix Distortion for Nucleotide Excision Repair
RNA pol II stalls at a damaged base on the transcribed strand
DDB1-DDB2 recognizes lesions on either DNA strand
XPC-HR23 is then recruited
Ubiquitylation of DDB2 and XPC may mediate the hand-off of the lesion to XPC-HR23
from Chu and Yang, Cell 135 , 1172 (2008)
Nucleotide Excision Repair in Human Cells
Mutations in at least seven XP genes inactivate nucleotide excision repair and cause xeroderma pigmentosum
The only pathway to repair thymine dimers in humans is nucleotide excision repair
XPC recognizes damaged DNA Helicase activities of XPB and XPD of TFIIH create sites for XPF and XPG cleavage
NER works mainly on helix-distorting damage caused by environmental mutagens
An oligonucleotide containing the lesion is released and the gap is filled by POL or and sealed by LIG1
from Lindahl and Wood, Science 286, 1897 (1999)
Transcription-coupled Repair
Repair of the transcribed strand of active genes is corrected 5-10-fold as fast as the nontranscribed strand
All the factors required for NER are required for transcription-coupled repair except XPC
The arrest of POL II progression at a lesion served as a damage recognition signal
Recruitment of NER factors also involves CS-A and CS-B
Nucleotide Excision Repair Pathway in Mammals
Cockayne’s Syndrome and Trichothiodystrophy are multisystem disorders defective in transcription-coupled DNA repair
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 of 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