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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
guanine
8-oxoguanine
from Banerjee et al., Nature 434, 612 (2005)
Repair of 8-oxo-G
Replication of the 8-oxoG strand
preferentially mispairs with A
and mimics a normal base pair
and results in a G-to-T transversion
8-oxoguanine DNA glycosylase/
b-lyase (OGG1) removes 8-oxo-G
and creates an AP site
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 A
HX 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
BER works primarily on modifications
caused by endogenous agents
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
DNA Glycosylases
from Xu et al., Mech.Ageing Dev. 129, 366 (2008)
Each glycosylase has limited substrate specificity, but there is redundancy in damage recognition
Mechanism of hOGG1 Action
from David, Nature 434, 569 (2005)
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
Nucleotide excision repair mainly works on helix distortion
and damage caused by environmental mutagens
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
from Chu and Yang, Cell 135 (1172 (2008)
Ubiquitylation of DDB2 and
XPC may mediate the hand-off
of the lesion to XPC-HR23
Nucleotide Excision Repair in Human Cells
NER works mainly on helix-distorting
damage caused by environmental mutagens
The only pathway to repair thymine dimers
in humans is nucleotide excision repair
Mutations in at least seven XP genes
inactivate nucleotide excision repair
and cause xeroderma pigmentosum
XPC recognizes damaged DNA
Helicase activities of XPB and XPD of
TFIIH create sites for XPF and XPG cleavage
An oligonucleotide containing the
lesion is released and the gap is filled
by POL d or e 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
Mismatch repair in E. coli
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 Eukaryotes
MutS homologs recognize mismatch
and form a ternary complex with
MulL homologs and the mismatch
PMS2 is a mismatch-activated strandspecific nuclease, and the break is directed
to the strand contain the preexisting nick
EXO1 excises the mismatch
The gap is filled in by PCNA, Pold and DNA ligase
Defective mismatch repair is the primary
cause of certain types of human cancers
from Hsieh and Yamane, Mech.Ageing Dev. 129, 391 (2008)
Causes of and Responses to ds Breaks
DSBs result from exogenous insults
or normal cellular processes
DSBs result in cell cycle
arrest, cell death, or repair
Repair of DSBs is by
homologous recombination
or nonhomologous end joining
from van Gent et al., Nature Rev.Genet. 2, 196 (2001)
Initiation of Double-stranded Break Repair
MRN complex recognizes
DSB ends and recruits ATM
ATM phosphorylates H2A.X and
recruits MDC1 to spread gH2A.X
TIP60 and UBC13 modify H2A.X
MDC1 recruits RNF8
which ubiquitylates H2A.X
RNF168 forms ubiquitin
conjugates and recruits BRCA1
from van Attikum and Gasser, Trends Cell Biol. 19, 204 (2009)
ATM Mediates the Cell’s Response to DSBs
DSBs activate ATM
ATM phosphorylation of p53,
NBS1 and H2A.X influence cell
cycle progression and DNA repatr
from van Gent et al., Nature Rev.Genet. 2, 196 (2001)
Repair of ds Breaks by Homologous Recombination
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
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)
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
from van Gent et al., Nature Rev.Genet. 2, 196 (2001)
Translesion DNA Synthesis
Replicative polymerase encounters
DNA damage on template strand
Catalytic site of replicative polymerases
is intolerant of misalignment between
template and incoming nucleotide
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
from Sale et al., Nature Rev.Mol.Cell Biol. 13, 141 (2012)
There are Multiple TLS Polymerases
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
from Sale et al., Nature Rev.Mol.Cell Biol. 13, 141 (2012)
Most mutations caused by DNA lesions
are caused by TLS polymerases
TLS Polymerases Can Be Accurate or Error-prone
Pol k bypasses an abasic site and often causes a -1 frameshift
Pol h bypasses a thymine dimer and inserts AA
Pol i is accurate with dA template and error-prone with dT template
Replicative polymerases insert dC or dA opposite 8-oxo-G, Pol i 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
AID deaminates dC to dU
Uracil DNA glycosylase forms
an abasic site, and REV1
incorporates dC opposite the site
MMR proteins lead to the formation of a
ss gap, PCNA is ubiquitylated, and Pol h
is recruited, generating mutations at A-T
from Sale et al., Nature Rev.Mol.Cell Biol. 13, 141 (2012)