Lecture 25: DNA Mutation, Proofreading, and Repair

Lecture 25: DNA mutation, Lecture Outline 11/2/05 proofreading, and repair • Review DNA replication machine

G • Fidelity of replication and proofreading C A T T A • Replicating the ends of chromosomes 1 nm

G C 3.4 nm C G • Mutation A T C G – Types of mutations T A T A – Repair mechanisms A T

A T

G C 0.34 nm A T

Figure 16.7a, c (c) Space-filling model 1 2

Replication overview DNA Polymerase III • Look at animations on your textbook CD • A complex enzyme with many subunits • one part adds the nucleotides • another helps it slide along the template • Look again at the animation from DNAi • another checks for mis-pairing – http://www.dnai.org – (go to the section on copying the code)

3 4 Figs. from http://www.mun.ca/biochem/courses/3107

1 Proofreading Fidelity of replication

• Even though bases preferentially pair G-C and A-T, Replication step error rate the initial error rate is about 1 in 10,000. 5䈊䊲㻖䈊㻃polymerization 1 × 105 • Many polymerases have “proofreading” ability. They 3䈊䊲㻘䈊 proofreading 1 × 102 can excise an mis-paired base and try again. Strand-directed mismatch repair 1 × 102 • This reduces the error rate to about 1 in a billion.

One polymerase subunit adds nucleotides Total error rate 1 × 109

Another “edits” out incorrec5t bases 6

What happens to the lagging strand The ends of eukaryotic chromosomal DNA get at the end of the chromosome? shorter with each round of replication 5′ End of parental Leading strand DNA strands Lagging strand 3′

Leaves a gap when the RNA Last fragment Previous fragment

primer is removed RNA primer 5′ 3’ Lagging strand 3′ Primer removed but Removal of primers and cannot be replaced replacement with DNA with DNA because where a 3′ end is available no 3′ end available for DNA polymerase 5′ 3′ 5’ Second round If they get short enough, of replication essential genes will 5′ eventually be deleted New leading strand 3′ New lagging strand 5′ 3′ Further rounds of replication Shorter and shorter Figure 16.18 daughter molecules 7 8

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

• Telomeres contain hundreds of simple tandem repeats. Carries its own RNA • In humans, the repeat sequence is TTAGGG template TTAGGG TTAGGG TTAGGG TTAGGG TTAGGG ...... • Cell lines with active telomerase live longer than those without telomerase. Extends the old – That may be important in allowing cancer cells to continue to (template) strand divide.

Normal synthesis of new DNA 9 10

Various kinds of mutations:

Mutations and repair Purine -> Purine or Pymimidine -> Pyrimidine: common

Purine -> Pymimidine: rare

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3 Types of base pair substitutions and mutations. Mutations can be caused by:

• Chemical mutagens • Ionizing radiation • Slippage during DNA replication • Spontaneous errors

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Chemical changes in one of the nucleotide bases UV damage (e.g. pyrimidine dimers) After replication, new strand has an A

Deamination changes C to U --U--- --T--- --A--- --A--- --C--- --U--- --G--- --G--- --G--- --C---

UV radiation can cause thymine dimers

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4 • In nucleotide excision repair – Enzymes cut out and replace damaged stretches of DNA

1 A thymine dimer distorts the DNA molecule. 2 A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed. Nuclease

DNA 3 Repair synthesis by polymerase a DNA polymerase fills in the missing nucleotides.

DNA ligase 4 DNA ligase seals the Free end of the new DNA To the old DNA, making the strand complete. Figure 16.17 17 18

Certain bacterial mutations Mismatch repair cause increased mutation rates Here is a mis-paired base that must be Defect in: Rifr mutants per 108 cells repaired: Wild-type (mut+ ) 5-10 G Pol III proofreading 4000-5000 T (mutD) Mis-match repair 760 How is the mistake recognized? (mutS) Base excision repair 8200 How does the mismatch repair system (mutY mutM) know which strand is the new one and which strand is the old one?

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5 Certain enzymes detect the MutS/L/H deformed helix that results from the incorrect pairing

G CH3 T The old (template) DNA G has methyl groups in GATC certain places G

MutS/L/H Re-synthesize DNA from the DNA pol I/III template using the normal DNA CH DNA Ligase polymerases G 3 T GATC CTAG CH3 G GATC Cut the newly C CTAG synthesized strand here Corrected base pair 21 22

• Various similar mechanisms for other types of mutations