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Lecture 25: DNA mutation, proofreading, and repair

Figure 16.7a, c (c) Space-filling model

C

T

A

A

T

CG

GC

A

C G

AT

AT

A T

TA

C

TA0.34 nm

3.4 nm

G

1 nm

G

T

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Lecture Outline 11/2/05

• Review DNA replication machine

• Fidelity of replication and proofreading

• Replicating the ends of chromosomes

• Mutation– Types of mutations– Repair mechanisms

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Figure 16.13

New strand Template strand5 end 3 end

Sugar A TBase

C

G

G

C

A

C

T

PP

P

OH

P P

5 end

Pyrophosphate

Phosphate

DNA synthesis goes 5’ to 3’

• DNA polymerases, add nucleotides to the 3 OH at the end of a growing strand

Nucleosidetriphosphate

OH

P

4Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Model for the “replication machine,” or replisome

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Replication overview• Look at animations on your textbook CD

• Look again at the animation from DNAi– http://www.dnai.org– (go to the section on copying the code)

6Figs. from http://www.mun.ca/biochem/courses/3107

DNA Polymerase III• A complex enzyme with many subunits• one part adds the nucleotides• another helps it slide along the template• another checks for mis-pairing

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Proofreading

• Even though bases preferentially pair G-C and A-T, the initial error rate is about 1 in 10,000.

• Many polymerases have “proofreading” ability. They can excise an mis-paired base and try again.

• This reduces the error rate to about 1 in a million.

One polymerase subunit adds nucleotides

Another “edits” out incorrect bases

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Fidelity of replication

Replication step error rate

5′→3′ polymerization 1 × 105

3′→5′ proofreading 1 × 102

Strand-directed mismatch repair 1 × 102

Total error rate 1 × 109

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What happens to the lagging strand at the very end of the chromosome?

3’

5’

Leaves a gap when the RNA primer is removed

5’

3’

3’

5’

10Figure 16.18

End of parentalDNA strands

Leading strandLagging strand

Last fragment Previous fragment

RNA primer

Lagging strand

Removal of primers andreplacement with DNAwhere a 3 end is available

Second roundof replication

New leading strand

New lagging strand 5

Further roundsof replication

Shorter and shorterdaughter molecules

5

3

5

3

5

3

5

3

3

Primer removed butcannot be replacedwith DNA becauseno 3 end available

for DNA polymerase

The ends of eukaryotic chromosomal DNA get shorter with each round of replication

If they get short enough, essential genes will eventually be deleted

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Telomerase

Carries its own RNA template

Extends the old (template) strand

Normal synthesis of new DNA

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What happens to the lagging strand that the end of the chromosome?

• Telomeres contain hundreds of simple tandem repeats. • In humans, the repeat sequence is TTAGGG

TTAGGG TTAGGG TTAGGG TTAGGG TTAGGG . . . . . . .

Lots of junk, so if the ends get slightly shorter, no essential genes are lost

• Cell lines with active telomerase live longer than those without telomerase. – That may be important in allowing cancer cells to continue to divide.

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Mutations and repair

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Various kinds of mutations:Purine -> Purine or Pymimidine -> Pyrimidine: common

Purine -> Pymimidine: rare

Some mutations change the code to a new amino acid

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Types of base pair substitutions and mutations.

Additions and deletions

Others are silent

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Mutations can be caused by:

• Chemical mutagens

• Ionizing radiation

• Slippage during DNA replication

• Spontaneous errors

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--C-----G---

--U-----A---

--U-----G--- --G---

--C---

--T-----A---

Deamination changes C to U

After replication, new strand has an A

Chemical changes in one of the nucleotide bases

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UV damage (e.g. pyrimidine dimers)

UV radiation can cause thymine dimers

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20Figure 16.17

Nuclease

DNApolymerase

DNAligase

A thymine dimerdistorts the DNA molecule.1

A nuclease enzyme cutsthe damaged DNA strandat two points and thedamaged section isremoved.

2

Repair synthesis bya DNA polymerasefills in the missingnucleotides.

3

DNA ligase seals theFree end of the new DNATo the old DNA, making thestrand complete.

4

• In nucleotide excision repair– Enzymes cut out and replace damaged

stretches of DNA

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Certain bacterial mutations cause increased mutation rates

Defect in: Rifr mutants per 108 cells

Wild-type (mut+ ) 5-10

Pol III proofreading

(mutD)

4000-5000

Mis-match repair

(mutS)

760

Base excision repair

(mutY mutM)

8200

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Mismatch repairHere is a mis-paired base that must be

repaired:

GT

How does the mismatch repair system know which strand is the new one and which strand is the old one?

How is the mistake recognized?

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GT

MutS/L/H

GT GATC

CTAG

CH3

MutS/L/H

The old (template) DNA has methyl groups in certain places

Certain enzymes detect the deformed helix that results from the incorrect pairing

Cut the newly synthesized strand here

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GGATC G

CH3

CH3

GC

GATCCTAG

DNA pol I/IIIDNA Ligase

Re-synthesize DNA from the template using the normal DNA polymerases

Corrected base pair

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• Various similar mechanisms for other types of mutations