Chen Yonggang

Zhejiang Univ. School of Medicine

Research Building C-616

Chenyg9@163.com

A DNA Repair Overview

Excellent Review Articles• Friedberg, EC (2003) DNA damage and

repair. Nature 421:436-440.

• Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S (2004) Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 73: 39-85.

Importance of Repair• DNA is the only biological macromolecule that

is repaired. All others are replaced.

• More than 100 genes are required for DNA repair, even in organisms with very small genomes.

• Cancer is a consequence of inadequate DNA repair.

DNA can be damaged by a variety of processes

1, Some spontaneously: deamination C U2, Others catalyzed by enviromental agentsa) UV light: dimerb) Chemicals: (1) deaminating agents (2) alkylating agentsc) Oxidative damage: hydrogen peroxide, hydroxyl radicals, superoxide radicals

Spontaneous base loss:

Several thousand purines and serval thousand pyrimidines per haploid genome per day!

AP Site: apyrimidinic site

or apurinic site

Spontaneous deamination:

~100 uracils per haploid genome per day.Also:Adenine to hypoxanthineGuanine to xanthine5-methyl cytosine to thymine

“Reactive Oxygen Species” (ROS) include O•, O-O•, HOOH, •OH

Thymine Thymine Glycol

Spontaneous production of 3-Methyl Adenine by S-Adenosylmethionine:

Several hundred per haploid genome per day!

Effects of Sunlight:(photodamage)

Cyclobutane pyrimidine dimers (CPDs)

T-T>T-C, C-T>C-C

DNA helix bends 7-9°

Effects of Sunlight:(photodamage)

Pyrimidine (6-4) pyrimidone photoproducts (6-4PPs)

T-C>C-C>T-T>C-T

DNA helix bends 44°

Some Additional Types of Damage:

• Replication errors

• Intra- and inter-strand crosslinks

• DNA-protein crosslinks

• Strand breaks

Types of DNA Repair

Direct repair(Direct reversal of damage)Excision repair(Excision of damaged

region, followed by precise replacement)

Recombination repair(strand break repair)

Damage bypass

An Example of Direct Repair: “Photoreactivation”

MTHF or 8-HDF

FADH-

Additional Examples of Direct Repair

• 6-4 photolyases

• Ligation of nicks

Excision Repair

Takes advantage of the double-stranded (double information) nature of the DNA molecule.

Mismatch repairBase excision repairNucleotide excision repair

Mismatch repair in E. coli

• Excision by UvrD (Helicase II and single-strand exonuclease)

• Gap filling by Polymerase I(in E. coli); Ligation by DNA ligase

Base Excision Repair

Base Excision Repair

• Several variations, depending on nature of damage, nature of glycosylase, and nature of DNA polymerase.

• All have in common the following steps:1. Removal of the incorrect base by an appropriate DNA

N-glycosylase to create an AP site.2. An AP endonuclease nicks on the 5’ side of the AP

site to generate a 3’-OH terminus.3. Extension of the 3’-OH terminus by a DNA

polymerase.

An example of a DNA N-glycosylase:

Pinch-push-pull mechanism suggested by crystal structures of glycosylases.

Some DNA N-glycosylases

have AP lyase

activity.

Initial steps of base-excision repair

Final steps of base-excision repair (DNA polymerase β pathway; short patch repair

Final steps of base-excision repair (replication pathway)

Nucleotide Excision Repair

• Extremely flexible• Corrects any damage that distorts the DNA molecule• In all organisms, NER involves the following steps:

1. Damage recognition

2. Binding of a multi-protein complex at the damaged site

3. Double incision of the damaged strand several nucleotides away from the damaged site, on both the 5’ and 3’ sides

4. Removal of the damage-containing oligonucleotide from between the two nicks

5. Filling in of the resulting gap by a DNA polymerase

6. Ligation

S. cerevisiae protein Human protein Probable function

Rad4 XPC GGR (also required for TC-NER in yeast); works with HR23B; binds damaged DNA; recruits

other NER proteins

Rad23 HR23B GGR; cooperates with XPC (see above); contains ubiquitin domain; interacts with proteasome and

XPC

Rad14 XPA Binds and stabilizes open complex; checks for damage

Rpa1,2,3 RPAp70,p32,p14 Stabilizes open complex (with Rad14/XPA)

Ssl2 (Rad25) XPB 3' to 5' helicase

Tfb1 GTF2H1 ?

Tfb2 GTF2H4 ?

Ssl1 GTF2H2 Zn finger; DNA binding?

Tfb4 GTF2H3 Ring finger; DNA binding?

Tfb5 TFB5; TTD-A Stabilization of TFIIH

Rad3 XPD 5' to 3' helicase

Tfb3/Rig2 MAT1 CDK assembly factor

Kin28 Cdk7 CDK; C-terminal domain kinase; CAK

Ccl1 CycH Cyclin

Rad2 XPG Endonuclease (3' incision); stabilizes full open complex

Rad1 XPF Part of endonuclease (5' incision)

Rad10 ERCC1 Part of endonuclease (5' incision)

Proteins Required for Eukaryotic Nucleotide Excision Repair

Nucleotide Excision Repair

Early Stages of Global Genome Repair

Initial Steps of Transcription-Coupled NER

Final Steps of Eukaryotic NER

Some of the proteins required for eukaryotic NERS. Cerevisiae Human Protein Probable functionRad 4 XPC GGR (also required for TC-NER in yeast; works with

HR23B; binds damaged DNA; recruits other NER proteins

Rad 23 HR23B GGR: cooperates with XPC; contains ubiquitin domain; interacts with proteasome and XPC

Rad 14 XPA Binds and stabilizes open complex; checks for damageRpa1, 2, 3 RPA p70, p32, p14 Stabilizes open complex (with Rad14/XPA)Ssl2 (Rad25) XPB 3’ to 5’ helicaseTfb1 GTF2H1 ?Tfb2 GTF2H4 ?Ssl1 GTF2H2 Zn Finger; DNA binding?Tfb4 GTF2H3 Ring Finger; DNA binding?Tfb5 TFB5; TTD-A Stabilization of TFIIHRad3 XPD 5’ to 3’ helicaseTfb3 MAT1 CDK assembly factorKin28 Cdk7 CDK; C-terminal domain kinase; CAKCcl1 CycH CyclinRad 2 XPG Endonuclease (3’ incision); stabilizes full open

complexRad1 XPF Part of endonuclease (5’ incision)Rad10 ERCC1 Part of endonuclease (5’ incision)

NER and Human Genetic Diseases• Xeroderma pigmentosum

1. Severe light sensitivity2. Severe pigmentation irregularities3. Frequent neurological defects4. Early onset of skin cancer at high incidence5. Elevated frequency of other forms of cancer

• Cockayne’s syndrome1. Premature aging of some tissues2. Dwarfism3. Light sensitivity in some cases4. Facial and limb abnormalities5. Neuroligical abnormalities6. Early death due to neurodegeneration

• Trichothiodystrophy1. Premature aging of some tissues2. Sulfur deficient brittle hair3. Facial abnormalities4. Short stature5. Ichthyosis (fish-like scales on the skin)6. Light sensitivity in some cases

Mitchell, Hoeijmakers and Niedernhofer (Divide and conquer: nucleotide excision repair battles cancer and ageing. Current Opinion in Cell Biology 15:232-240, 2003).

Recombinational Repair

Strand-break repair

• Usually essential for cell survival• Many pathways, whose relative importance varies

between and within organisms Double-strand break repair by homologous recombination

(HR) Double-strand break repair by non-homologous end joining

(NHEJ) Single-strand break repair (SSBR)

Homologous Recombination is Based on the Ability of Single DNA Strands to Find Regions of Near-

Perfect Homology Elsewhere in the Genome

• Facilitation of Homology Searching by RecA and its Eukaryotic Homologs

• Eukaryotic proteins important in this process include Rad51, Rad52, Rad54, Rad55, Rad57, Rad59. BRCA1 and BRCA2 interact with Rad51 and may regulate it.

Bypass synthesis corrects defect occuring in replication

• Bypass polymerases with reduced fidelity can read through lesions, increasing the possiblity of inducing errors in the new DNA

• Such enzymes have low processivity, only synthesizing short fragments and limiting copy errors

• Excision repair can cut out damaged nucleotides and repair damage