Chapter 29/30

DNA Mutation and Repair

Pages 975-978 AND 1009-1012

All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777

Learning objectives: Understand the following

• what are the different types of mutation• how are mutations introduced into DNA• what are the different types of repair machinery• how does each repair mechanism function• how is human disease associated with DNA repair defects

Define a Mutation

• A mutationA mutation is a stable change in DNA structure

heritableheritable Base sequence Base sequence is changedis changed

• Mutations are classified into several “types”Mutations are classified into several “types”

Molecular Nature of Mutation(what types of mutations arise)

• Point mutationsPoint mutations (base substitutions): one base substituted for another

• Transition mutations = Ato G or C to T (purine to purine or pyrimidine to pyrimidine)

• Transversion mutations = purine to pyrimidine or vice versa

• Can arise from mispairing, insertion of base analogs, or chemical mutagens (nitrous acid, hydroxylamine and alkylating agents)

Consequences of Point mutations

• A missensemissense mutation: This results in one wrong codon and one wrong amino acid.

Consequences of Point mutations

A nonsensenonsense mutation: If the change in the base sequence results in a stopstop codon, the protein would be terminated at that point in the message.

Consequences of Point mutations

• A sensesense mutation: This occurs when the change in the DNA sequence results in a new codon still coding for the same amino acid.

Other types of mutations

• Insertions and deletionsInsertions and deletions: result in frameshift mutations (less common)

Insert a G here

DNA Mutation - How?

1) Through mistakes during DNA replication

2) Spontaneous mutations (deamination, depurination that occur naturally)

3) Induced mutations caused by environmental agents (chemical mutagens, UV radiation)

Errors Arising Due to base MispairingDuring DNA Replication (page 975)

Tautomerization1)

Syn vs anti conformation (flipping around glycosidic bind)

2)

H2O acting as a bridge

3)

Bases can mispair during DNA replication through three (quite rare) mechanisms that change the H-bonding properties of the base so it is incorporated incorrectly.

Base AnalogsBase Analogs may get incorporated during DNA replication

= thymine analoggets incorporated duringDNA replication opposite an A

frequent tautomer change-now pairs with G

Compounds that chemically resemble a nucleotide base closely enough that during DNA replication, they can be incorporated into the DNA in place ofthe natural base.

•The base analog, 2-AP pairs with T.

•But 2-AP can also pair with C via a singe H-bond.

•Therefore, if 2-AP gets in DNA, then upon replication a C is sometimes inserted.

Another base analog

DNA Mutation - How? - continued

1) Through mistakes during DNA replication

2) Spontaneous mutations (deamination, depurination that occur naturally)

3) Induced mutations caused by environmental agents (chemical mutagens, UV radiation)

Spontaneous DNA Mutation

• Deamination:

- C to U (now pairs with T) (100/cell/day)

- A to Hypoxanthine (now pairs with C)

- G to Xanthine (still pairs with C)

• Depurination/Depyrimidination:

- removes purine base from DNA(104 bases /cell/day) or pyrimidine bases (less frequent)

• Oxygen radical damage

-breaks sugar rings

Oxidative deaminationThe primary amino groups of nucleic acid bases are somewhat unstable. They can be converted to keto groups in reactions like the one pictured here:

In a mammalian cell, ~100 uracils are generated per cell per day in this fashion. Other reactions include conversion of adenine to hypoxanthine, and guanine to xanthine.

Oxidative deaminationother examples

Spontaneous Base Loss

The glycosyl bond linking DNA bases with deoxyribose is labile under physiological conditions.

Within a typical mammalian cell, several thousand purines and several hundred pyrimidines are spontaneously lost per cell per day.

Loss of a purine or pyrimidine base creates an apurinic/apyrimidinic (AP) site (also called an abasic site)

Oxygen radical damageOxygen radical damage

DNA Mutation - How?- continued

1) Through mistakes during DNA replication

2) Spontaneous mutations (deamination, depurination that occur naturally)

3) Induced mutations caused by environ-mental agents (chemical mutagens, UV radiation)

Mutation through Exposure to Environmental Agents

• Chemical mutagens:

- Nitrous acid (HN02) formed from nitrites (in preserved meats) reacting with stomach acid, causes oxidative deamination

- Alkylating agents add methyl or ethyl groups to bases and change their pairing properties

- Intercalating agents (ethidium bromide, acridine orange) distort the helix causing frameshift insertions or deletions.

Nitrous acid

Agents causing oxidative deamination

Nitrosamines

Agents causing alkylation

The first report of mutagenic action of a chemical was in 1942 by Charlotte Auerbach, who showed that nitrogen mustard (component of poisonousmustard gas used in World Wars I and II) could cause mutations in cells.

Intercalating Agents

Mutation through Exposure to Environmental Agents

• Ultraviolet Radiation: classified in terms of its wavelength

•UV-C (180-290 nm)--"germicidal"--most energetic and lethal, it is not found in sunlight because it is absorbed by the ozone layer

•UV-B (290-320 nm)--major lethal/mutagenic fraction of sunlight

•UV-A (320 nm--visible)--"near UV"--also has deleteriouseffects (primarily because it creates oxygen radicals) but it produces very few pyrimidine dimers.

Tanning beds have UV-A and UV-B.

• Ultraviolet Radiation:

- causes dimers to form between adjacent T

bases on a strand of DNA (or between adjacent T-C bases, hence the term pyrimidine dimer)

- a cyclobutyl ring links the adjacent T’s

- this interferes with the ability of the T’s to

base pair to the opposite strand, and blocks DNA replication

The most frequent photoproducts are bonds formed between adjacent pyrimidines within one strand

The most frequent are CPDs, cyclobutane pyrimidine dimers

T-T CPDs are formed most readily, followed by T-C or C-T; C-C dimers are least abundant.

CPDs cause a distortion in the DNA chain structure. The two adjacent pyrimidines are pulled closer to each other than in normal DNA.

T-T CPD withcyclobutyl ring in blue

DNA Repair

A fundamental difference from RNA, protein, and lipid

• All these others can be replaced, but DNA must be preserved

• We have seen how changes get stably incorporated into DNA

• How do the mutations get repaired??• E. coli is the model system for understanding different

repair mechanisms

• Direct repair - fixes pyrimidine dimers (UV damage)

• Excision repair:

i) Base Excision Repair (BER) - fixes abnormal bases (uracil, hypoxanthine, alkylated bases)

ii) Nucleotide Excision Repair (NER) - fixes large structural changes and helix distortions (pyrimidine dimers, bulky base adducts)

• Mismatch repair - fixes mismatches

Mechanisms of DNA Repair

Pyrimidine dimers can be repaired by: DNA photolyase:

•Uses energy from light absorption•Contains chromophores (light absorbing agents)•Action spectrum is blue/near UV light range

Steps in the repair mechanism:1) Enzyme recognizes and binds to the damage2) Light absorption by chromophore converts it to an excited state3) Chromophore donates an electron to the cyclobutyl dimer4) Dimer is destabilized and undergoes a series of electron rearrangements which result in monomeric pyrimidines

Direct Repair SystemRepairs damage without removing the damaged base

Direct Repair SystemRepairs damage without removing the damaged base

Photolyase contains 2 chromophores that absorb light energy. In all photolyases, one of the chromophores is FADH-, and the other is either methenyl-tetrahydrofolate (MTHF) or 8-hydroxy-5-deazaflavin (8-HDF). MTHF and 8-HDF act as primary light gatherers (green in the diagram), transferring their energy to FADH- (yellow). The energy from FADH- is used to split the dimer

Direct Repair SystemRepairs damage without removing the damaged base

• CPD photolyases are found in bacteria, fungi, plants and many vertebrates, but not in placental mammals.

• As we shall see, humans use a different repair pathwayto fix pyrimidine dimers caused by UV light

Excision Repair

• Excision repair:

i) Base Excision Repair (BER) - fixes abnormal bases (uracil, hypoxanthine, alkylated bases)

ii) Nucleotide Excision Repair (NER) - fixes large structural changes and helix distortions (pyrimidine dimers, bulky base adducts)

Base Excision Repair

• Abnormal bases are recognized by a set of DNA glycosylaseDNA glycosylase enzymes, each one recognizing a specific abnormal base

• For example, uracil glycosylase recognizes uracil in DNA

• After recognizing the damage the DNA glycosylase binds to it and initiates a repair pathway by hydrolyzing off the base

A DNA glycosylasebinds to damaged base

AP endonuclease binds and cleaves the DNA backbone

Exonuclease removes a segment of DNA

DNAP fills in the gap

Ligase seals the nick

Cleaves N-glycosidic bondNote that the AP siteIs identical to that made by spontaneousdepurination or depyrimidination.

1. Removal of the incorrect base by an appropriate DNA glycosylase to create an AP site

2. Nicking of the damaged DNA strand by AP endonuclease upstream of the AP site, thus creating a 3'-OH terminus adjacent to the AP site

3. Excision of the AP site, followed by extension of the 3'-OH terminus by a DNA polymerase

Steps in Base Excision Repair

Base Excision Repair is not Enough

• BER can’t deal with all types of damage since it requires a DNA glycosylase to recognize each specific damage.

• The huge variety of chemical mutagens combined with radiation and oxygen radicals make too many types of damage to each be recognized by a different DNA glycosylase.

Nucleotide Excision Repair

• Fortunately, a different, more flexible damage repair mechanism called NERNER has evolved.

• NER machinery uses a limited number of proteins to recognize damaged regions in DNA based on their abnormal structureabnormal structure as well as on their abnormal chemistryabnormal chemistry, then excises and replaces them.

In all organisms, NER has the same steps:

• Damage recognition • Binding of a protein complex at the damaged site • Double incision of the damaged strand several

nucleotides away from the damaged site, on both the 5' and 3' sides

• Removal of the damage-containing fragment from between the two nicks

• Filling in of the resulting gap by a DNA polymerase

• Ligation

• Fixes damage causing large distortions in the helical structure (e.g. UV-induced pyrimidine dimers, other base adducts)

• In E. coli the NER machinery repairing UV damage is a protein complex:

UvrA + UvrB + UvrC = ABC excinucleaseexcinuclease

• UvrD helicase, DNAP I, and ligase also participate

Nucleotide Excision Repair

• 2 UvrA proteins form a complex with one UvrB protein in an ATP-dependent reaction

• The complex recognizes UV damage by distortion in the helix

• The UvrA proteins dissociate from the complex after ATP hydrolysis.

• This leaves UvrB bound across from the damage

Nucleotide Excision Repair in E. coli

• Now UvrB can recruit UvrC protein to the complex

• UvrC activates UvrB to nick the DNA 4 ntds 3’ from the pyrimidine dimer

• Then UvrB activates UvrC to nick the DNA 7 ntds 5’ from the pyrimidine dimer

• This leaves a fragment of DNA containing the damage that can now be removed

Nucleotide Excision Repair in E. coli

• A helicase, UvrD, uses ATP hydrolysis to power the unwinding of the damaged DNA fragment. This reomoves UvrC

• The gap in the DNA is now filled in by DNAPI or II, reomving uvrB in the process

• Finally, DNA ligase seals the nick

Nucleotide Excision Repair in E. coli

• Critical for repairing UV-induced damage

(because we don’t do direct repair)• The principal is the same as in bacteria:

i.e. damage is recognized, an excinuclease makes a gap, DNAP fills the gap

• But the proteins are different (20-30)

• Defects in NER proteins cause genetic disorders

NER in Humans

NER in HumansXeroderma pigmentosum (XP)

Genetic disorder with symptoms:

-extreme sensitivity to sunlight (by ~age 2),

and >1000X higher risk of skin cancer

(by ~age 8)

Defect is in repair of UV damage

Gene mapping identified several

repair proteins (called XP proteins)

XP-C and XP-A recognize pyrimidine dimers

XP-B and XP-D have helicase activity

XP-G and XP-F have nuclease activity

NER in Humans

XP-C recognizes pyrimidine dimers

XP-B and XP-D are helicases thatseparate the DNA strands aroundthe damage. RPA keeps the strands apart

XP-G and XP-F are endonucleases that cut the DNA on either side ofthe damage

XP-A binds to the pyrimidine dimerand helps to recruit other proteinsto a complex

The cut fragment is removed and the gap is filled in by DNAP or

Real-world biochemistryIn 1997, NASA developed a Prototype UV garment for children with XP, Porphyria and other sun Related Disorders to have a Quality of life and Freedom. This NASA UV Protective Project designed for XP was completed in 1998 and the UV garments are being supplied to children of parents that have requested them.

Mismatch Repair • Corrects mismatchesmismatches after DNA replication

• Increases replication fidelity by 102-103 fold

• Mismatch repair systems scan newly-replicated DNA duplexes for mismatched bases

• PROBLEM: which of the 2 strands has the correct sequence and which has the error?

Mismatch Repair • Since the parentparent (template) strand should

have the correct sequence, repair machinery identifies the parent strand and copies it to correct the mistake

• Since methylation occurs post-replication, repair proteins identify methylated strand methylated strand as parentas parent, remove mismatched bases on other strand and replace them

Steps in Mismatch Repair

• MutSMutS recognizes mismatches and binds to them. Binding of MutLMutL stabilizes the complex.

• The MutS-MutL complex activates MutHMutH, which locates a nearby methyl group and nicks the newly synthesized strand opposite the methyl group.

• A helicase (UvrDUvrD) unwinds from the nick in the direction of the mismatch, and a single-strand specific exonuclease cuts the unwound DNA

• the gap is filled in by DNAP III and sealed by DNA ligase.

Repair of Mismatch Replication ErrorsRepair of Mismatch Replication Errors(in E. coli)(in E. coli)

1.

2.

3.

4.

1. MutSMutS recognizes mismatches and binds to them. Binding of MutLMutL stabilizes the complex.

2. The MutS-MutL complex activates MutHMutH, which locates a nearby methyl group and nicks the newly synthesized strand opposite the methyl group.

Continued from the previous slide1.

2.

3.

4.

3. A helicase (UvrDUvrD) unwinds from the nick in the direction of the mismatch, and a single-strand specific exonuclease cuts the unwound DNA

4. The gap is filled in by DNAP III and sealed by DNA ligase.

Repair of Mismatch Replication ErrorsRepair of Mismatch Replication Errors(in E. coli)(in E. coli)

Eukaryotic Mismatch Repair • Detailed mechanism is unclear

• Identification of the parent strand is different

• Human proteins were found similar in sequence to MutL and MutS

• called hMutL (MLH) and hMutS (MSH)

• Defects in either MLH or MLS result in an Defects in either MLH or MLS result in an enhanced susceptibility to cancerenhanced susceptibility to cancer (HNPCC=hereditary non-polyposis colon cancer)

We have now finished Chapters 29 and 30

All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777

For next class please read:

Chapter 31Pages 1014-1023