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

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

Free dNTPs are much more susceptible to oxidative damage than bases in duplex DNA

Oxidized precursors are misincorporated and are mutagenic

MTH1 removes oxidized nucleotides from the pool

from Dominissini and He, Nature 508, 191 (2014)

MTH1 Prevents Incorporation of Oxidized dNTPs into DNA

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)

Inhibition of MTH1 Selectively Kills Cancer Cells

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-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

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)

UV-induced pyrimidine dimers

Nucleotide Excision Repair

Bulky adducts

Repairs helix-distorting lesions

Intrastrand crosslinks

ROS-generated cyclopurines

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)

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)

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

XPG nuclease binds to the complex

RPA protects the undamaged strand from nucleases

NER – Lesion Verification

from Marteijn et al., Nature Rev.Mol.Cell Biol. 15, 465 (2014)

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)

PCNA recruits DNA polymerase to fill ss gap

Nick is sealed by DNA ligase

NER – Gap Filling and Ligation

from Marteijn et al., Nature Rev.Mol.Cell Biol. 15, 465 (2014)

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

Chromatin Dynamics in GG-NER

from Marteijn et al., Nature Rev.Mol.Cell Biol. 15, 465 (2014)

Clinical Implications of Defective NER

GG-NER is elevated in germ cells to maintain the entire genome to prevent mutagenesis

TC-NER is elevated in somatic cells to repair expressed genes to prevent cell death

Defective GG-NER increases cancer predisposition

Defective TC-NER causes premature cell death, neurodegeration and accelerates aging

Xeroderma pigmentosum

Cockayne Syndrome

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 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)

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