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
from David et al., Nature 447, 941 (2007)

4. MTH1 Prevents Incorporation of Oxidized dNTPs into DNA
Free dNTPs are much more
susceptible to oxidative damage
than bases in duplex DNA
Oxidized precursors are
misincorporated and are mutagenic
from Dominissini and He, Nature 508, 191 (2014)
MTH1 removes oxidized
nucleotides from the pool

5. Inhibition of MTH1 Selectively Kills Cancer Cells
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)

6. UV-Irradiation Causes Formation of Thymine Dimers
from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-38

7. Nonenzymatic Methylation of DNA
Formation of 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

8. 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

9. Repair Pathways for Altered DNA Bases
from Lindahl and Wood, Science 286, 1897 (1999)

10. 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

11. 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

12. 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

13. 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

14. Nucleotide Excision Repair
Repairs helix-distorting lesions
UV-induced pyrimidine dimers
Bulky adducts
Intrastrand crosslinks
ROS-generated cyclopurines

15. 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)

16. 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)

17. NER – Lesion Verification
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
RPA protects the undamaged
strand from nucleases
XPG nuclease binds to the complex
from Marteijn et al., Nature Rev.Mol.Cell Biol. 15, 465 (2014)

18. 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)

19. NER – Gap Filling and Ligation
PCNA recruits DNA polymerase to fill ss gap
Nick is sealed by DNA ligase
from Marteijn et al., Nature Rev.Mol.Cell Biol. 15, 465 (2014)

20. Chromatin Dynamics in GG-NER
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
from Marteijn et al., Nature Rev.Mol.Cell Biol. 15, 465 (2014)

21. Clinical Implications of Defective NER
GG-NER is elevated in germ cells to maintain the entire genome to prevent mutagenesis
Defective GG-NER increases cancer predisposition
Xeroderma pigmentosum
TC-NER is elevated in somatic cells to repair expressed genes to prevent cell death
Defective TC-NER causes premature cell death, neurodegeration and accelerates aging
Cockayne Syndrome

22. Mismatch Repair
Repairs DNA replication errors and insertion-deletion loops
Decreases mutation frequency by
Plays a role in triplet repeat expansion, somatic
hypermutation and class switch recombination

23. Mismatch repair in E. coli
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)

24. 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)

25. 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)

26. Chromatin Modification at DSB
from Panier and Boulton, Nature Rev.Mol.Cell Biol. 15, 7 (2014)
MRN complex recognizes DSB, and recruits ATM which phosphorylates H2A.X
gH2A.X is recognized by MDC1 which recruits more MRN and ATM
ATM phosphorylates MDC1 which recruits RNF8
RNF8 ubiquitylates a substrate (X) which recruits RNF168
53BP1 is recruited by H4K20me2 and H2AK13/15Ub

27. 53BP1 is not Recruited to Undamaged Chromatin
from Panier and Boulton, Nature Rev.Mol.Cell Biol. 15, 7 (2014)
H4K20me2 is a common histone modification and is not limited to DSB sites
H4K20me2 is masked in undamaged chromatin and cannot recruit 53BP1

28. Ubiquitylation by Chromatin Remodelling
Cascade Allows 53BP1 Recruitment
H4K20me2 is unmasked in the presence
of DSB by ubiquitylation enzymes
from Panier and Boulton, Nature Rev.Mol.Cell Biol. 15, 7 (2014)

29. The Choice Between HR and NHEJ
BRCA1 and 53BP1 binding to
DSB chromatin is antagonistic
H4 modification controls loading of 53BP1
During G1, 53BP1 binds to chromatin,
RIF1 inhibits BRCA1 association, end
resection is limited, and NHEJ is promoted
During S and G2, CDK phosphorylates CtIP,
promotes BRCA1 association, allows
extensive end resection, and promotes HR
from Panier and Boulton, Nature Rev.Mol.Cell Biol. 15, 7 (2014)

30. Rearrangement of Chromatin Around the DSB
gH2A.X is exchanged for H2A.Z
H2A.Z promotes the formation of more
open chromatin and facilitates repair
from Volle and Dalal, Curr.Opin.Genet.Dev. 25, 8, 2014

31. 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)

32. 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

33. 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)

34. 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)

35. 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)

36. 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

37. 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

38. 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)