DNA Damage

We, as well as our genetic complement do not live in a vacuum. Our DNA is, on a daily basis, confronted with numerous molecules and forces (originating form both endogenous and exogenous sources) that have the capability of altering DNA structure.

It makes sense that proper maintenance of organismal homeostasis requires the maintenance of genomic homeostasis. Increased incidence of unrepaired lesions in DNA will ultimately render DNA useless as an information molecule either by making the DNA untranscribable or untranslatable, or by introducing deleterious mutations.

“The intrinsic chemical lability of the DNA molecule poses a formidable threat to its persistence…” Dr. Philip Hanawalt

General Types of DNA Damage

Oxidation

Free radicals arising from oxidative metabolism can readily damage DNA. Most common damage is oxidation of cytosine to uracil. Walker, page 8

Many compounds can alkylate DNA. Such modifications can range from the addition of simple methyl (-CH3) groups to longer alkyl chains.

Alkylation

Alkylating agents are most reactive with nucleophilic centers present in DNA.

Fig 1-32 in Walker, page 32

Alkylating agents can be either monofunctional (single reactive group) or bifunctional (multiple reactive groups).

Nitrosoureas are a class of simple alkylating agents.

3HC N C N NO2 N O

NH2

N-Methyl-Nʼ-Nitro-N-nitrosoguanidine (MNNG)

Bifunctional alkylating agents can form bonds at two nucleophilic centers. Nitrogen mustards and sulfur mustards (mustard gas) are in this class of compounds.

Owing to their bi-functional nature these compounds can form either intrastrand or interstrand crosslinks.

Nitrogen mustard

Bulky DNA adducts

Many compounds can place bulky, non organic groups within DNA.

Cis-Platinum is an example of this class of compounds

Pt

H3N

H3N

Cl

Cl

Cis-platin can wreak all kinds of havoc with DNA...

walker page 34

Psoralen

Walker page 36 Color plate 6

UV-induced damage Exposure of DNA to UV light leads to the formation of UV-induced photoproducts

Two major photoproducts:

Cyclobutane Pyrimidine Dimers (CPD)

“Thymine dimers”

4,6 photoproducts

handout

NH

N

O

O

H3C NH

N

O

O

H3C

cyclobutane dimer

4,6-photoproduct

NN

HO

O

H3C

NN

O

O

H3C H

H

OH

Mechanisms of DNA Repair The cell has evolved a complex set of mechanisms For repairing damaged DNA:

•  Direct repair • Photolyases • Methylguanine Methyltransferase (MGMT)

• Excision Repair • Base Excision Repair (BER) • Mismatch Repair (MMR) • Nucleotide Excision Repair (NER)

• Global Genome NER • Transcription-coupled NER

• Strand Break Repair •  Recombination-based repair •  Illegitimate recombination repair •  Non-homologous end joining (NHEJ)

Direct Repair of Alkylation Damage

O6-Methylguanine Methyltransferase (MGMT)

• repairs O6-methylguanine adducts

• enzyme transfers a methyl group from the O6 position of guanine to a cysteine residue in the protein, thereby repairing the damage by direct reversal

• suicide enzyme- consumed in the reaction it catalyzes

• conserved from E. coli to human

Direct Repair of UV damage- Photolyase •  Photoreactivation

–  Repairs T-T cyclobutane dimers with visible light •  Binds T-T dimers w/ high specificity - no light required •  Breaking the dimers requires light --> an electron transfer reaction

–  Requires 2 cofactors - FADH2 and MTHF –  Not found in placental mammals

Excision Repair

Base Excision Repair (BER) Generally involved in repair of simple types of base damage (ie, base oxidation)

Mismatch Repair (MMR) Generally involved in repair of mismatched bases arising due to errors in replication.

Nucleotide Excision Repair (NER) Generally repairs more complex types of DNA lesions that result in inhibition of DNA replication or RNA transcription (ie UV-induced photoproducts).

All of the Excision Repair mechanisms follow the same general biochemical sequence of events in DNA repair:

1) recognition of lesion 2) removal of lesion 3) resynthesis of DNA 4) ligation of free DNA ends

DNA Damage Repaired by BER Oxidative Damage

NH

NN

N

O

NH

HO

H

NH

NN

N

O

NH

HHO

8-oxoguanine major

8-hydroxyguanine

NH

NHN

HN

O

NH2

O

H

formamidopyrimidine (FaPy)

NH

N

O

O

H3C

HO

HO

thymine glycol

Alkylation Damage

N

NN

N

NH2

CH3

NH

NN

N

O

NH2

H3C

N

NNH

N

N

3-methyladenine 7-methylguanine 1,N6-ethenoadenine

Base Excision Repair (BER)

Errors Repaired by Mismatch Repair •  Specificity of mismatch repair

–  G•T, A•C > G•G, A•A > T•T, C•T, G•A >> CC –  Correlated with the frequency of replication errors

•  DNA polymerases tend to make G•T and A•C mispairs most frequently

•  Repairs insertions and deletions (I/D’s) –  1, 2, and 3 base I/D’s repaired efficiently –  4 base I/D’s repaired marginally –  5 base I/D’s not repaired at all

•  Polymerase frameshift mutations give rise to I/D’s –  Occur most frequently in repetitive sequences or homopolymeric runs

5’--A A A 3’--T T T T G C--5’

5’--A A 3’--T T T T G C--5’

A 5’--A A A A C G--3’ 3’--T T T T G C--5’

A

5’--A A A 3’--T T T T G C--5’

5’--A A A 3’--T T T G C--5’

T

5’--A A A C G--3’ 3’--T T T G C--5’

T

1 base insertion

1 base deletion

• The mismatch repair (MMR) system primarily corrects base-base mismatches and short generated as a consequence of DNA replication errors

Mismatch Repair

nick nick

Global Genome Nucleotide Excision Repair in humans

Transcription-coupled Nucleotide Excision Repair in humans

Cancer and DNA repair

Cancer is a disease of the genome

Lost or relaxed genomic stability allows the accumulation of mutations which fuel cancer development and progression

Commonly, DNA repair (and damage response mechanisms) are targeted for inactivation in cancers that arise sporadically

Some cancer-predisposition genetic disorders are recognized

HNPCC has a population prevalence of 1 in ~3,000 in the US population

• 18% of colorectal cancers under 45 years • 28% of colorectal cancers under 30 years

HEREDITARY NONPOLYPOSIS COLORECTAL CANCER

HNPCC LYNCH SYNDROME (the broader syndrome, includes ovarian, endometrial, gastric)

Mutations in the mismatch repair (MMR) system are present in ~70% of HNPCC families.

The MMR system is principally involved in the repair of replicative DNA errors.

Mutations in the MMR protein MLH1 account for ~ 50%, MSH2 ~40%. Mutation of other MMR proteins such as MSH6, PMS1, PMS2 have been reported.

HNPCC at the molecular level

Microsatellite Instability a hallmark feature associated with loss of MMR

PCR of the BAT-26 marker

Normal size of BAT-26 locus

Altered size of BAT-26 locus in tumor

Schneider et al (2000) Int. J. Oncol. 89:444

XERODERMA PIGMENTOSUM

XP patients display blistering or freckling on minimum sun exposure.

XP patients also show premature aging of skin, lips, eyes, mouth and tongue and have a significantly increased incidence of cancer in these same areas

Other manifestations include: blindness resulting from eye lesions or surgery for skin cancer close to the eyes

developmental disabilities

mental retardation

high frequency hearing loss, progressing to deafness

The chief clinical hallmark of XP is sunlight-induced skin cancers (>1000-fold increased risk of skin cancer).

UV-irradiation results in the formation of cyclobutane pyrimidine dimers (thymine dimers) and 4,6 photoproducts in DNA

Most UV-induced lesions are repaired by global-genome nucleotide excision repair (GG-NER).

Many of the molecules necessary for GG-NER were discovered in the study of XP.

CPD 4-6 photoproduct

M1 and M2 are two separate recessive mutations that both result in the same phenotype

Fuse cells and test for complementation of phenotype

Mutant phenotype

Mutant phenotype

Mutant phenotype

Identifying genes based on complementation grouping