Chemistry and Biochemistry 153B DNA, RNA, and Protein Synthesis Fall 2007 - Albert Courey

Powerpoint #4: DNA Damage and Repair

4.2) DNA damage

A gallery of horrors
UV damage Hydrolytic and oxidative deamination General oxidation Alkylation

4.3) UV damage

So stay out of the sun because its dangerous.

4.4) Hydrolytic or oxidative deamination

Nitrosonium ion Electron hungry Formed from: nitrates and nitrites (common food preservatives) Also formed from nitrosamines (byproducts of rubber production)

So processed foods are dangerous. But so is water!

4.5) The problem with uracil in DNA

Deamination of C to U occurs at a rate of 10-7/day (~100 times in each human cell per day).

5 A 3

C G

A U

T A

G U

G C

3 5

Due to incorporation of dUTP by polymerase

Due to spontaneous deamination of C->U

If U was a normal component of DNA, there would be no way to know if a U is supposed to be there, or if it arose by the deamination of a C residue

4.6) Oxidative Damage

Cytochrome C oxidase Examples of DNA oxidative damage G ROS T
ROS ROS

8-oxo-G 5-formyl-T
ROS

deoxyribose

ribose

Neutralization of ROS
2O2-+2H+ 2H2O2 Superoxide disumutase Catalase H2O2 +O2

ROS can also directly attack and break the DNA backbone

So heavy breathing is dangerous! 2H2O+ O2

No cellular neutralization of hydroxide radical

4.7) Unusual templating properties of 8-oxo G

5 3 X + dNTP Pol I N X X is either G or 8-oxoG N is either A or C Rate of reaction measured as a function of dNTP concentration

!!!
From Hsu et al (2004) Nature 431, 217-221.

Two rounds of DNA replication result in GC to TA transversion GC ROS 8-oxoG:C 8-oxoG:A T:A

The most common mutation associated with cancer!!!

Question: Why do DNA polymerases incorporate A opposite 8-oxoG? See problem 4 on Problem set #2.

4.8) Alkylation
Any nucleophilic atom on the DNA (e.g., N7, N6, N3, O2, O6, etc.) can be alkylated sometimes leading to changes in basepairing specificity. For example, alkylation of guanine O6 can lead to GC to AT transition mutations.

4.9) Some favorite alkylating agents

Ethylmethane sulfonate (EMS) - a favorite among geneticists

MNNG - another tool of geneticists - a nitrosamine Alkyl groups transferred are shown in red Nitrogen mustard

Mustard gas
Cl-

4.10) How to minimize damage to your DNA Avoid Avoid Avoid Avoid Avoid Avoid chemical warfare processed foods the highways sunlight aerobic activities water

But if all these precautions fail, our cells have multiple DNA repair pathways to undo the damage!!!

4.11) Some DNA repair strategies

Direct reversal of the damage Base excision repair Nucleotide excision repair SOS repair Double-strand Break Repair Recombination repair
Will discuss briefly Read about it in your book - vv. 117677. You are expected to know this material even though I wont lecture on it. You can read about them in your book, but you wont be held responsible for these topics.

4.12) An example of direct reversal: MGMT

Warning: note the errors in these Lewis structures

Can also remove ethyl group Not an enzyme, since it is irreversibly modified by the reaction In humans, mutations in the gene encoding MGMT are linked to several forms of familial cancer. So this reaction must play an important role in tumor suppression. In E. coli, the alkylated form of this enzyme acts as a transcription factor to induce the expression of its own gene as well as genes encoding other repair proteins. So MGMT serves as a sensor of the alkylation state of the DNA.

4.13) Base excision repair pathway

X DNA Glycosylase
OH

X = damaged base (e.g., uracil, hypoxanthine, 8-oxoguanine, etc.)

AP endonuclease
OH Pi OH

There are many dozen such enzymes each one recognizing a different X (e.g., uracil-N-glycosylase)

5 -> 3 exonuclease
OH Pi

In bacteria, the exonuclease and polymerase functions are both provided by Pol I through the process of nick translation

DNA polymerase DNA ligase

Lets look at a couple of the glycosylases.

4.14) Uracil DNA glycosylase - base recognition

The base is flipped out

Basis for specificity Asn residue makes H-bond contacts with W/C face of flipped out U Tyr residue sterically interferes with 5-methyl, so T is excluded from active site

4.15) 8-oxoguanine DNA glycosylase base recognition

Base is flipped out

Basis for specificity Gln residue makes H-bond contacts with W/C face of flipped out 8-oxoG Specificity for 8-oxoG over G is provided by H-bond between Gly carbonyl O and N7

4.16) What you should know from slides 4.2 to 4.15

I) DNA damage A. B. C. II) A. B. How do UV, oxidation, alkylation, and hydrolysis lead to DNA damage? Why is it important for DNA to contain thymine instead of uracil? How does DNA damage affect the templating properties of the bases? Direct reversal - how does MGMT reverse alkylation and how does it serve as a sensor of the overall alkylation state of the genome? Base excision repair 1) 2) C. What is the overall strategy of base excision repair? How do DNA glycosylases recognize their cognate damaged bases?

Repair strategies

Nucleotide excision repair - What is the overall strategy of nucleotide excision repair and what kind of lesions is it used to repair? Read vv. 1173-1178, 1286-1287