1. Transcription Course: Molecules to Cells Lecturer: David Mu DNA Repair

2. Most DNA Damages Are Repaired Less than 1 in 1000 accidental base changes in DNA results in a permanent mutation. Many human diseases are characterized by a compromised DNA repair capacity:

3. DNA Undergoes Spontaneous Changes Hydrolytic attack – blue arrows Oxidative damage – red arrows Methylation – green arrows

4. Spontaneous Depurination and Deamination Cytosine to uracil in DNA occurs at a rate of about 100 bases per cell per day.

5. UV Light Can Produce Pyrimidine Dimers T<>T Primary repair pathway Bacteria and yeast: DNA photolyase Human: Nucleotide excision repair

6. Consequences of Unrepaired DNA Damages 1.Replication stoppage or infidelity 2.Transcription blockage 3.Cell cycle arrest 4.Mutant gene products (RNA and/or proteins) 5.Genome instability Fate of DNA Damages 1.Unrepaired 2.Repaired 3.Mis-repaired 4.Inducing cell death 5.May be fixed in DNA

7. How Common Is Replication Mis-Incorporation? Haploid size = 3300 Megabase pairs = 3.3 x 10 9 (= billion) base pairs Diploid size = double that Misincorporation (10 -5 ) x not proofread (10 -2 ) x escape mismatch repair (10 -3 ) = 10 -10 Thus, less than one replication error is fixed per cell division

8. DNA Repair Mechanisms

9. Direct Chemical Reversal of DNA Damages PL=photolyase methenyltetrahydrofolate (MTHF) flavin adenine dinucleotide (FAD) FAD FADH 2

10. Direct Chemical Reversal of DNA Damages Photolyase Mechanism Not present in human cells

11. Mismatch Repair (MMR) - Bacterial Modrich 2015 E. coli mismatch repair is dictated by the state of adenine methylation at d(GATC) sequences. Because this modification occurs after DNA synthesis, newly synthesized DNA exists transiently in an unmodified state, and it is this transient absence of methylation that directs repair to the new strand.

12. Mismatch Repair (MMR) - Bacterial Modrich 2015 Keywords: Bidirectional

13. Mismatch Repair (MMR) – Bacterial (MutH) Modrich 2015 MutS is responsible for mismatch recognition. MutL is recruited to the MutS–heteroduplex complex in an ATP-dependent reaction, but does not otherwise alter the covalent nature of the helix. Assembly of the MutL–MutS–heteroduplex ternary complex leads to dramatic activation of this latent MutH d(GATC) endonuclease A system comprised of MutH, MutL, MutS, UvrD (DNA helicase II), exonuclease I (Exo I), SSB, DNA polymerase III holoenzyme, and DNA ligase was sufficient to reconstitute methyl-directed mismatch repair in vitro.

14. Mismatch Repair (MMR) - Bacterial Modrich 2015 MutS is responsible for mismatch recognition. MutL is recruited to the MutS–heteroduplex complex in an ATP-dependent reaction, but does not otherwise alter the covalent nature of the helix. Assembly of the MutL–MutS–heteroduplex ternary complex leads to dramatic activation of this latent MutH d(GATC) endonuclease A system comprised of MutH, MutL, MutS, UvrD (DNA helicase II), exonuclease I (Exo I), SSB, DNA polymerase III holoenzyme, and DNA ligase was sufficient to reconstitute methyl-directed mismatch repair in vitro.

15. Mismatch Repair (MMR) - Human A strand break serves as a signal for human DNA mismatch repair Modrich 2015 No repair in the continuous DNA strand !!!

16. Mismatch Repair (MMR) - Human MutLα endonuclease in mismatch-provoked excision Modrich 2015 Human mismatch recognition factor: MutS 

17. Mismatch Repair (MMR) - Human Modrich 2015

18. Human DNA Mismatch Repair - homework In the last paragraph of his 2015 Nobel lecture, Paul Modrich speculated on the strand signal of human DNA mismatch repair. READ IT !!!! Mechanisms in E. coli and Human Mismatch Repair (Nobel Lecture). Modrich P. Angew Chem Int Ed Engl. 2016 Jul 18;55(30):8490-501. 2016 May 20. Review. PMID: 27198632 (in Blackboard)

19. Base Excision Repair (BER)

20. Nucleotide Excision Repair (NER) in Bacteria Sancar 2015 Important To Remember

21. Transcription-coupled NER in Bacteria

22. Nucleotide Excision Repair (NER) in Humans A disease in humans known as Xeroderma Pigmentosum XP is a rare inherited disease of humans which, among other things, predisposes the patient to pigmented lesions on areas of the skin exposed to the sun and an elevated incidence of skin cancer. It turns out that XP can be caused by mutations in any one of several genes - all of which have roles to play in NER. James Cleaver went around and collected cells from hundreds of these patients. He then figured out that the disease was made up of eight genes named XP-A through XP-G plus one called XP-V for variant.

23. There are 8 XP complementation groups XP-A participates in photoproduct recognition and DNA binding This binding may be followed by the formation of a quasi-stable complex consisting of XPA, XPC, human single- strand binding protein (RPA/HSSB), and TFIIH, which then acts as a nucleation site for binding of the incision/excision enzymes. XP-B is a 3’-> 5’ DNA helicase that may be involved in unwinding the DNA 5'-ward of a damaged base XP-C is a single-stranded DNA binding protein that is essential for repair of the nontranscribed regions of the genome, that acts in the initial step of damage recognition. XP-D is a 5'–3' helicase, a component of transcription factor TFIIH may be involved in 3'-ward unwinding of the DNA in the vicinity of a damaged base

24. There are 8 XP complementation groups XP-E is thought to be involved with the recognition of damaged DNA because it has the capacity to bind to UV- damaged DNA XP-F in association with the ERCC1 protein, incises DNA on the 5' side of the damaged site XP-G incises DNA 3' to the damaged site XP-V protein is a low-fidelity class Y DNA polymerase, that can replicate UV-induced pyrimidine dimers in vivo with the insertion of the correct bases in the daughter strand

25. Cell-Based Complementation Groups of XP + Fuse two XP cells from different patients together Fused cell UV irradiate Plate cells and check survival resistant sensitive

26. Basal transcription factor consists of DNA repair proteins Google.comSchultz et al. 2000 Xeroderma Pigmentosum (XP) patient XPB = Xeroderma Pigmentosum complementation group B XPD = Xeroderma Pigmentosum complementation group D

27. In Vitro DNA Repair Assays - I

28. In Vitro DNA Repair Assays - II Nucleotide Excision Assay

29. Mechanism of Human Excision Nuclease Sancar 2015

30. Other Repair Mechanisms - recombination Albert et al.

32. Questions 1.What are the major types of DNA damage repaired by each of the following pathways: (a) photoreversal, (b) base excision repair, (c) nucleotide excision repair, and (d) mismatch repair? 2.Specify which of the repair pathways (a, b, c, and/or d) listed in the previous question is associated with each of the following enzymes, protein complexes or gene products: AP endonuclease, hMSH2, DNA photolyase, MutH, XPA, uracil DNA glycosylase, DNA ligase, TFIIH, DNA polymerase. 3.True or False – Only the initial steps in DNA repair are catalyzed by enzymes that are unique to the repair process; the later steps are typically catalyzed by enzymes that play more general roles in DNA metabolism. 4.Discuss the following statement: The DNA repair enzymes that correct damage introduced by deamination and depurination must preferentially recognize such defects on newly synthesized DNA strands.