from Marnett and Plastaras, Trends Genet' 17, 214 2001

1
Endogenous DNA Damage
from Marnett and Plastaras, Trends Genet. 17, 214
(2001)
2
Biological Molecules are Labile
RNA is susceptible to hydrolysis
Reduction of ribose to deoxyribose gives DNA
greater stability
N-glycosyl bond of DNA is more labile
DNA damage occurs from normal cellular operations
and random interactions with the environment
3
Spontaneous Changes that Alter DNA Structure
deamination
oxidation
depurination
from Alberts et al., Molecular Biology of the
Cell, 4th ed., Fig 5-46
4
Hydrolysis of the N-glycosyl Bond of DNA
from Alberts et al., Molecular Biology of the
Cell, 4th ed., Fig 5-47
Spontaneous depurination results in loss of
10,000 bases/cell/day
Causes formation of an AP site not mutagenic
5
Deamination of Cytosine to Uracil
from Alberts et al., Molecular Biology of the
Cell, 4th ed., Fig 5-47
Cytosine is deaminated to uracil at a rate of
100-500/cell/day
Uracil is excised by uracil-DNA-glycosylase to
form AP site
6
Deamination of A and G Occur Less Frequently
A is deaminated to HX base pairs with C
G is deaminated to X base pairs with C
from Alberts et al., Molecular Biology of the
Cell, 4th ed., Fig 5-52
7
5-Methyl Cytosine Deamination is Highly Mutagenic
Deamination of 5-methyl cytosine to T occurs
rapidly - base pairs with A
5-me-C is a target for spontaneous mutations
from Alberts et al., Molecular Biology of the
Cell, 4th ed., Fig 5-52
8
Oxidative Damage of DNA
Oxidative damage results from aerobic metabolism,
environmental toxins, activated macrophages, and
signaling molecules (NO)
Compartmentation limits oxidative DNA damage
9
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)
10
Consequences of Guanine Oxidation to 8-Oxoguanine
Action of 8-oxoguanine DNA glycosylase/b-lyase
(hOGG1) creates an AP site
Replication of the oxoG strand preferentially
mispairs with A
from Bruner et al., Nature 403, 859 (2000)
11
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
12
UV-Irradiation Causes Formation of Thymine Dimers
from Lodish et al., Molecular Cell Biology, 6th
ed. Fig 4-38
13
Nonenzymatic Methylation of DNA
Formation of 600 3-me-A residues/cell/day are
caused by S-andnosylmethionine
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
14
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
15
Spores Use Strategies to Overcome Intrinsic
Instability of DNA
DNA exists in an A-like conformation and is bound
to proteins that reduce the rate of depurination
Lack of dNTPs in spores prevents DNA repair
before germination
Extensive DNA repair occurs upon spore germination
16
Repair Pathways for Altered DNA Bases
from Lindahl and Wood, Science 286, 1897 (1999)
17
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
18
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
19
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
20
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)
21
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
22
Nucleotide Excision Repair Pathway in Mammals
Cockaynes Syndrome and Trichothiodystrophy are
multisystem disorders defective in
transcription-coupled DNA repair
23
Mismatch Repair in Human Cells
MSH2 and MSH6 bind to mismatch- containing DNA
and distinguish between the template and newly
synthesized strand
MLH1 nicks the newly synthesized DNA and an
exonuclease removes the mismatched base
The gap is filled in by DNA polymerase and DNA
ligase
Defective mismatch repair is the primary cause
of certain types of human cancers
from Lodish et al., Molecular Cell Biology, 6th
ed. Fig 4-37
24
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)
25
ATM Mediates the Cells Response to DSBs
DSBs activate ATM
ATM phosphorylation of p53, NBS1 and H2AX
influence cell cycle progression and DNA repatr
from van Gent et al., Nature Rev.Genet. 2, 196
(2001)
26
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
27
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)
28
Structure of Rad50 and the Mre11 Complex
Mre11 complex maintains genome integrity and
participates in HR and NHEJ
AT-like syndrome and Nijmegen breakage syndrome
are caused by mutation in MRE11 or NBS1
Dimerization domain of Rad50 keeps DNA fragments
within close proximity
from de Jager and Kanaar, Genes Dev. 16, 2173
(2002)
29
XRCC4
X-ray-repair-cross-complementing defective repair
in Chinese hamster mutant 4
30
The SCID Mouse is Defective in DSB Repair
The SCID mouse carries a mutation preventing the
production of mature B and T cells
The phenotype is a defect in the development of
the immune system and hypersensitivity to
ionizing radiation
The phenotype is caused by a mutation in DNA-PK
31
Translesion Replication by DNA Polymerase V
Translesion DNA synthesis occurs in the absence
of Pol III
Translesion DNA polymerases are error prone and
exhibit weak processivity
Most of the mutations caused by DNA damaging
agents are caused by TLR
TLR protects the genome from gross rearrangements
Pol V is regulated by LexA and the SOS response
from Livneh, J.Biol.Chem. 276, 25639 (2001)