Chapter 10 DNA damage and repair

1. Defects in repair cause disease

2. Common types of DNA damage

3. DNA repair pathwaysDirect repairBase excision repairNucleotide excision repairMismatch repairRecombination repairSOS response

DNA Damage

• The vast majority of DNA damage affects the primary structure of the double helix

• Occurs at a rate of 1,000 to 1,000,000 molecular lesions per cell per day, only 0.000165% of the human genome's approximately 6 billion bases (3 billion base pairs)

DNA repair defects cause disease

Damage from where?

• Consequences of DNA replication errors

• Chemical agents acting on the DNA

• UV light imparting energy into DNA molecule

• Spontaneous changes to the DNA

Common types of DNA damage -- 1

1. Depurination （脱嘌呤） : A, G2. Deamination （脱胺作用） : C --> U,

A --> Hypoxanthine （次黄嘌呤）

06_25_mutations.jpg

DNA level view of the same two events as last slide

Common types of DNA damage -- 2

Pyrimidine dimers 嘧啶二聚体 (UV induced).

Common types of DNA damage -- 3

Two carcinogens that mutate (the P53 gene) by base alkylation 烷化

+ Mismatches (mistakes in DNA synthesis)Interstrand cross-links, Double-strand DNA breaks

Total damage from all mechanisms: 104 - 106 lesions/day!

DNA repair

• Damaged DNA must be repaired

• If the damage is passed on to subsequent generations, then we use the evolutionary term - mutation. It must take place in the germ cells - the gametes - eggs and sperm

• If damage is to somatic cells (all other cells of the body bar germ cells) then just that one individual is affected.

Why repair DNA?

• DNA pol does a great job, but not good enough• Introduces errors in about 1 in 10E7 nucleotides

added, which it does not correct• Other mechanisms exist (as we will see) to correct

many of the errors left by the replication system• Most mistakes and damage corrected (99% -leaving

just a few - only 1 in 10E9 errors are left)• Mutations are permanent changes left in the DNA

Diverse DNA repair systems

General mechanisms shared in eukaryotes

1. Direct repair, 直接修复 e.g. pyrimidine dimers

2. Base excision repair 碱基切除修复 3. Nucleotide excision repair 核苷酸切除修复 4. Mismatch repair 错配修复 5. Recombination repair 重组修复 6. SOS response （ SOS 反应）

• Two thymines connected together by UV light.

• Photoreactivation (bacteria, yeast, some vertebrates - not humans)

DNA photolyase

Diverse DNA repair systems

•General mechanisms shared in eukaryotes1. Direct repair, 直接修复 e.g. pyrimidine dimers

2. Base excision repair 碱基切除修复 3. Nucleotide excision repair 核苷酸切除修复 4. Mismatch repair 错配修复 5. Recombination repair 重组修复 6. SOS response （ SOS 反应）

Base excision repair

Base excision repair pathway (BER).(a) A DNA glycosylase recognizes a damaged base and cleaves between the base and deoxyribose in the backbone.

(b) An AP endonuclease cleaves the phosphodiester backbone near the AP site.

(c) DNA polymerase I initiates repair synthesis from the free 3’ OH at the nick, removing a portion of the damaged strand (with its 5’3’ exonuclease activity) and replacing it with undamaged DNA.

(d) The nick remaining after DNA polymerase I has dissociated is sealed by DNA ligase.

AP= apurinic ( 脱嘌呤 ) or

apyrimidinic( 脱嘧啶 )(a=without)

Damaged base

A DNA glycosylase initiates base excision repair

Examples of bases cleaved by DNA glycosylases:Uracil (deamination of C)

8-oxoG paired with C (oxidation of G)

Adenine across from 8-oxoG (misincorporation)

Thymine across from G (5-meC deamination)

Alkyl-adenine (3-meA, 7-meG, hypoxanthine)

Damaged base

Diverse DNA repair systems

• Augment DNA polymerase proofreading• Mostly characterized in bacteria• General mechanisms shared in eukaryotes 1. Direct repair, e.g. pyrimidine dimers 2. Base excision repair 3. Nucleotide excision repair 4. Mismatch excision repair 5. Recombination repair

Nucleotide excision repair

(a) Two excinucleases (excision endonucleases) bind DNA at the site of bulky lesion. (b) One cleaves the 5’ side and the other cleaves the 3’ side of the lesion, and the DNA segment is removed by a helicase. (c) DNA polymerase fills in the gap and (d) DNA ligase seals the nick.

UvrA recognizes bulky lesions

UvrB and UvrC make cuts

UvrD

Structural distortion = signal

Nucleotide excision repair -- eukaryotes

Mutations in any of at least seven genes, XP-A through XP-G, cause an inherited sensitivity to UV-induced skin cancer called xeroderma pigmentosum. The XP proteins are among >30 required for nucleotide excision repair.

Two pathways of increasing complexity

Base Excisio

nrepair

Nucleotide Excisionrepair

Diverse DNA repair systems

• Augment DNA polymerase proofreading• Mostly characterized in bacteria• General mechanisms shared in eukaryotes

1. Direct repair, e.g. pyrimidine dimers

2. Base excision repair 3. Nucleotide excision repair 4. Mismatch excision repair -- replication errors 5. Recombination repair

Mismatch repair

Mut S binds mismatchMut L links S to H

Mut H recognizes the parental strand

Which strand is new and which is the parent?

Mismatch repair

Which strand is new and which is the parent? The mutation is in the new strand! -CH3 marks the parental strand!

MutH - Binds 7-meGATCMutS - Binds mismatchMutL - links MutH and MutS

Mismatch repair -- Recognition

Which strand is new and which is the parent? The mutation is in the new strand! A-CH3 marks the parental strand!

MutS - Binds mismatchMutL - links MutH and MutSMutH - Binds GmeATC

DNA is threaded through the MutS/MutL complex. The complex moves simultaneously in both directions along the DNA until it encounters a MutH protein bound at a hemimethylated GATC sequence. MutH cleaves the unmethylated strand on the 5’ side of the G in the GATC sequence.

Mismatch repair -- Resolution

1. The combined action of DNA helicase II, SSB, and one of many different exonucleases (only two are labeled) removes a segment of the new strand between the MutH cleavage site and a point just beyond the mismatch.

2. The resulting gap is filled in by DNA polymerase III, and the nick is sealed by DNA ligase.

Dam methylationDam methylationThe bacterial can discriminate between old and new replicated strands.

Old strands are methylated at adenine in -GATC- sequences by an enzyme called the Dam methylase.

So the mismatch on the newly replicated strand is preferentially excised.

Mismatch repair systems are present in human cells and defects lead to colon cancer.

Mismatch repair -- Hereditary Non-Polyposis Colon Cancer (HNPCC) gene (Humans)

遗传性非息肉病性结肠癌

HNPCC results from mutations in genes involved in DNA mismatch repair, including:

• several different MutS homologs• Mut L homolog• other proteins: perhaps they play the role of

MutH, but not by recognizing hemi-methylated DNA (no 6meA GATC methylation in humans, no dam methylase)

Diverse DNA repair systems

• Augment DNA polymerase proofreading• Mostly characterized in bacteria• General mechanisms shared in eukaryotes

1. Direct repair, e.g. pyrimidine dimers

2. Base excision repair 3. Nucleotide excision repair 4. Mismatch excision repair -- replication errors 5. Recombination repair

Recombinational RepairRecombinational Repair

The gap in the undamaged parental strand is filled by DNApol I and ligase. The thyminedimer can now be repaired by excision repair

5’

5’

3’

3’

3’5’

Thymine dimer 5’

5’

3’

3’

3’5’

Thymine dimer

5’

5’

3’

3’

3’5’

Thymine dimer

SOS response

• The SOS response is a global response to DNA damage in which the cell cycle is arrested and DNA repair and mutagenesis are induced.

• The SOS uses the RecA protein (Rad51 in eukaryotes). • During normal growth, the SOS genes are negatively

regulated by LexA repressor protein dimers .• Activation of the SOS genes occurs after DNA damage

by the accumulation of ssDNA regions generated at replication forks, where DNA polymerase is blocked.

The SOS responseThe SOS response

In response to extensive genetic damage there is a regulatory system that co-ordinates the bacterial cell response. This results in the increased expression of >30 genes, involved in DNA repair, these include:recA - activator of SOS response, recombinationsfiA (sulA) - a cell division inhibitor (repair before

replication)umuC, D - an error prone bypass of thymine dimers

(loss of fidelity in DNA replication)uvrA,B,C,D - excision repair

The SOS response is regulated by two key genes: recA & lexA

Gene mutation

• Mutations are heritable permanent changes in a genomic sequence.

• Mutation can be caused by – spontaneous （自发的） errors in DNA

replication or meiotic recombination ；– radiation, viruses, transposons and mutagenic

chemicals;– by the organism itself, by cellular processes

such as hypermutation.

Classification of mutation types • By effect on structure

– Small-scale mutations – Large-scale mutations in chromosomal structure

• By effect on function – Loss-of-function mutations – Gain-of-function mutations – Lethal mutations – A back mutation or reversion

• By effect on fitness– A harmful mutation – A beneficial mutation

• By impact on protein sequence – A frameshift mutation – A nonsense mutation – A Missense mutations – A neutral mutation

By impact on protein sequence

• A frameshift mutation

• A nonsense mutation

• A Missense mutations

• A neutral mutation

A frameshift mutation

• A frameshift mutation is a mutation caused by insertion or deletion of a number of nucleotides that is not evenly divisible by three from a DNA sequence

Mutations: Insertion

Normal gene

GGTCTCCTCACGCCA

↓

CCAGAGGAGUGCGGU

Codons

↓

Pro-Glu-Glu-Cys-Gly

Amino acids

Addition mutation

GGTGCTCCTCACGCCA

↓

CCACGAGGAGUGCGGU

↓

Pro-Arg-Gly-Val-Arg

A frame shift mutation

Mutations: Deletions

Normal gene

GGTCTCCTCACGCCA

↓

CCAGAGGAGUGCGGU

Codons

↓

Pro-Glu-Glu-Cys-Gly

Amino acids

Deletion mutation

GGTC/CCTCACGCCA

↓

CCAGGGAGUGCGGU

↓

Pro-Gly-Ser-Ala-Val

A frame shift mutation

A nonsense mutation

• A nonsense mutation is a point mutation in a sequence of DNA that results in a premature stop codon, or a nonsense codon in the transcribed mRNA DNA: 5' - ATG ACT CAC CGA GCG CGA AGC TGA - 3'shut

3' - TAC TGA GTG GCT CGC GCT TCG ACT –5'

mRNA: 5' - AUG ACU CAC CGA GCG CGA AGC UGA - 3' Protein: Met Thr His Arg Ala Arg Ser Stop

DNA: 5' - ATG ACT CAC TGA GCG CGA AGC TGA - 3'shut

3' - TAC TGA GTG ACT CGC GCT TCG ACT –5'

mRNA: 5' - AUG ACU CAC TGA GCG CGA AGC UGA - 3' Protein: Met Thr His Stop

A Missense mutations

• A missense mutation is a point mutation in which a single nucleotide is changed, resulting in a codon that codes for a different amino acid (mutations that change an amino acid to a stop codon are considered nonsense mutations, rather than missense mutations).

A Missense mutations

Normal gene

GGTCTCCTCACGCCA

↓

CCAGAGGAGUGCGGU

Codons

↓

Pro-Glu-Glu-Cys-Gly

Amino acids

Substitution mutation

GGTCACCTCACGCCA

↓

CCAGUGGAGUGCGGU

↓

Pro-Arg-Glu-Cys-Gly

A neutral mutation

• A neutral mutation is a mutation that has no effect on fitness.

• Many or even most mutations to noncoding DNA are neutral.

Point mutation: a single base change

CATGCACCTGTACCAGTACGTGGACATGGT

CATCCACCTGTACCAGTAGGTGGACATGGT

transition (T-A to C-G) transversion (T-A to G-C)

base pair substitutions转换 transition: pyrimidine to pyrimidine, purine to purine

颠换 transversion: pyrimidine to purine

normal sequenceCATTCACCTGTACCAGTAAGTGGACATGGT

Deletion/ insertion

CATCACCTGTACCAGTAGTGGACATGGT

deletionCATGTCACCTGTACCAGTACAGTGGACATGGT

insertion

deletions and insertions can involve one or more base pairs

normal sequenceCATTCACCTGTACCAGTAAGTGGACATGGT

Inversion

normal sequenceCATTCACCTGTACCAGTAAGTGGACATGGT

CATCTACCTGTACCAGTAGATGGACATGGT

The consequence of mutation

• Have no effect

• Alter the product of a gene

• Prevent the gene from functioning properly or completely

• About 70 percent of these mutations having damaging effects

06_19_sickle_cell.jpg

Consider…

• Sunlight - sunbathing or daily exposure– Impact of ozone depletion 臭氧层破坏– Impact on different skin tones

• Environmental degradation 劣化

Evolution acts on mutations

• If we did not have mutation then we would all be the same!

• Any changes in the environment would be deleterious to all members of the population equally

• But mutation does exist and it is supported by comparison of related organisms…

请大家自学突变剂的作用