Oxidative Damage of DNA

1
Oxidative Damage of DNA
Oxidative damage results from aerobic metabolism,
environmental toxins, activated macrophages, and
signaling molecules (NO)
Compartmentation limits oxidative DNA damage
2
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)
3
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
4
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
5
UV-Irradiation Causes Formation of Thymine Dimers
from Lodish et al., Molecular Cell Biology, 6th
ed. Fig 4-38
6
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
7
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
8
Repair Pathways for Altered DNA Bases
from Lindahl and Wood, Science 286, 1897 (1999)
9
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
10
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
11
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
12
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
13
Nucleotide Excision Repair
Nucleotide excision repair mainly works on helix
distortion and damage caused by environmental
mutagens
14
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)
15
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)
16
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
17
Nucleotide Excision Repair Pathway in Mammals
Cockaynes Syndrome and Trichothiodystrophy are
multisystem disorders defective in
transcription-coupled DNA repair
18
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
19
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)
20
Mismatch Repair in Eukaryotes
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, 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)
21
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)
22
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)
23
ATM Mediates the Cells 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)
24
Repair of ds Breaks by Homologous Recombination
ssDNAs with 3ends 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
25
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)
26
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)
27
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)
28
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
29
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
30
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)