1. 1 Lecture 25: DNA mutation, proofreading, and repair Figure 16.7a, c (c) Space-filling model C T A A T C G GC A C G A T A T AT T A C T A 0.34 nm 3.4 nm G 1 nm G T

2. 2 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

3. 3 Figure 16.13 New strandTemplate strand 5 end 3 end Sugar A T Base C G G C A C T P P 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 Nucleoside triphosphate OH P

4. 4 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Model for the “ replication machine, ” or replisome

5. 5 Replication overview Look at animations on your textbook CD Look again at the animation from DNAianimation from DNAi –http://www.dnai.orghttp://www.dnai.org –(go to the section on copying the code)

6. 6 Figs. 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

7. 7 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

8. 8 Fidelity of replication Replication step error rate 5′→3′ polymerization 1 × 10 5 3′→5′ proofreading1 × 10 2 Strand-directed mismatch repair 1 × 10 2 Total error rate1 × 10 9

9. 9 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’ 5’

10. 10 Figure 16.18 End of parental DNA strands Leading strand Lagging strand Last fragmentPrevious fragment RNA primer Lagging strand Removal of primers and replacement with DNA where a 3 end is available Second round of replication New leading strand New lagging strand 5 Further rounds of replication Shorter and shorter daughter molecules 5 3 5 3 5 3 5 3 3 Primer removed but cannot be replaced with DNA because no 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

11. 11 Telomerase Carries its own RNA template Extends the old (template) strand Normal synthesis of new DNA

12. 12 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.

13. 13 Mutations and repair

14. 14 Various kinds of mutations: Purine -> Purine or Pymimidine -> Pyrimidine: common Purine -> Pymimidine: rare Some mutations change the code to a new amino acid

15. 15 Types of base pair substitutions and mutations. Additions and deletions Others are silent

16. 16 Mutations can be caused by: Chemical mutagens Ionizing radiation Slippage during DNA replication Spontaneous errors

17. 17 --C--- --G--- --U--- --A--- --U--- --G--- --C--- --T--- --A--- Deamination changes C to U After replication, new strand has an A Chemical changes in one of the nucleotide bases

18. 18 UV damage (e.g. pyrimidine dimers) UV radiation can cause thymine dimers

19. 19

20. 20 Figure 16.17 Nuclease DNA polymerase DNA ligase A thymine dimer distorts the DNA molecule. 1 A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed. 2 Repair synthesis by a DNA polymerase fills in the missing nucleotides. 3 DNA ligase seals the Free end of the new DNA To the old DNA, making the strand complete. 4 In nucleotide excision repair –Enzymes cut out and replace damaged stretches of DNA

21. 21 Certain bacterial mutations cause increased mutation rates Defect in:Rif r mutants per 10 8 cells Wild-type (mut + )5-10 Pol III proofreading (mutD) 4000-5000 Mis-match repair (mutS) 760 Base excision repair (mutY mutM) 8200

22. 22 Mismatch repair Here is a mis-paired base that must be repaired: GTGT 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?

23. 23 GTGT MutS/L/H GTGT GATC CTAG CH 3 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

24. 24 G GATC G CH 3 GCGC GATC CTAG DNA pol I/III DNA Ligase Re-synthesize DNA from the template using the normal DNA polymerases Corrected base pair

25. 25 Various similar mechanisms for other types of mutations