1. BIMS 808 General and Molecular Genetics 4/4- 4/7- 4/9/2008 DNA Repair and the DNA damage response Pat Concannon Room 6056 Jordan Hall (mornings) Room 6111 Multistory building (afternoons) patcon@virginia.edu Resources: Cell Research Jan. 2008 Friedberg textbook, DNA Repair and Mutagenesis, ASM Press

2. DNA is, in fact, so precious and so fragile that we now know that the cell has evolved a whole variety of repair mechanisms to protect its DNA from assaults by radiation, chemicals, and other hazards. This is exactly the sort of thing that the process of evolution by natural selection would lead us to expect. -Francis Crick, 1990 Thus it is not inheritance and variation which brings about evolution, but the inheritance of variation, and this in turn is due the general principle of gene construction which causes persistence of autocatalysis despite the alteration in structure of the gene itself. -Hermann Muller, 1922

3. Some history.... Hugo de Vries, Principles of the theory of mutation. Science (1914) –First coined the term “mutation” –Idea that genetic variation arises from some unspecified “intracellular perturbation” Hermann J. Muller, Artificial transmutation of the gene, Science (1927) –X-ray mutagenesis of Drosophila –In a single experiment increased the known number of Drosophila mutants by 50%

4. More history... Delbruck’s target theory of gene inactivation—1935 UV induction of mutations--1941 –Maximal induction at 265nm wavelength Avery demonstrates that genes are constructed of DNA—1944 Kelner and Dulbecco separately discover that UV induction of mutations can be counter-acted by exposure to visible light

5. DNA damage induced by UV light Predominant lesion Thymine dimers are potent blocks to both DNA replication and transcription

6. Photoreactivation directly reverses DNA damage Employs a specific enzyme, a photolyase Photolyases common in bacteria and lower eukaryotes but entirely absent from placental mammals Contain two non- covalently associated chromophores

7. Even more history... 1950s –An E.coli strain hypersensitive to UV isolated –Liquid holding recovery independent of visible light 1960s –Loss of DNA fragments in bacteria recovering from UV –Multiple complementation groups of E. coli UV sensitive mutants –Repair synthesis in E. coli What about mammals (like humans)?

8. Xeroderma pigmentosum (XP) In humans, UV damage repaired only by nucleotide excision repair (NER) Defects in NER lead to at least 3 different genetic disorders: –XP –Cockayne syndrome –Trichothiodystrophy XP is characterized by skin lesions on sun exposes surfaces, frequent benign and malignant tumors and neurologic abnormalities

9. Magnitude of the DNA repair problem DNA in a typical mammalian cell is under constant insult just from endogenous sources....

10. The major mechanisms of repair for nucleotide damage in mammals are via excision Base Excision Repair (BER) –Repair of oxidizes, alkylated or inappropriate bases as well as abasic sites –Responsible for repair of the majority of DNA damage Nucleotide Excision Repair (NER) –Mechanism of DNA excision and repair synthesis that corrects damage caused by agents that create bulky DNA adducts (e.g. thymine dimers) –Most versatile of excision repair mechanisms Mismatch Repair (MMR) –Repairs base-base mismatches and insertion/deletion mispairings arising during DNA replication and recombination We’ll start with a discussion of lesions repaired by BER…

11. Alkylation: addition of ethyl or methyl groups to any nucleophilic atom on DNA Sugar In this example, alkylation on the 0 6 residue of guanine results in a change of base pairing C>T

13. Basic mechanism of BER Excision of damaged base by specific DNA glycosylase –Creates an abasic (AP) site AP endonuclease (APE) cleaves the AP site –Generates 3’OH and 5’ deoxyribose termini DNA polymerase fills the single nucleotide gap A DNA ligase seals the nick after synthesis

14. DNA glycosylases confer specificity on BER

15. Unifying feature of DNA glycosylases is that they scan for DNA damage by extrahelical flipping of damaged bases Lau. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 13573-13578

16. “Flipped” base must fit into a highly specific active site in the glycosylase Lau et al. (2000) Proc. Natl. Acad. Sci. USA 97, 13573-13578

17. Why doesn’t DNA have uracil? Deamination of cytosine to uracil occurs at a rate of ~10 -7 per day—this means about 100 events per cell per day U pairs well with either A or G, so this would lead to additional errors if not corrected

18. Glycosylases for oxidized bases are typically bifunctional with intrinsic AP lyase activity Glycosylases not essential in mammals while APE1 is Some ends require cleaning by one of a variety of enzymes to be compatible for closing Single Nucleotide (SN) BER involves simple insertion and ligation Long Patch BER utilizes enzymes involved in DNA replication –Typically 2-8 nucleotides are added

19. Distinct end-cleaning enzymes are utilized for different types of AP sites Mutations in Aprataxin result in Ataxia Oculomotor Apraxia (AOA1) a neurologic disorder

20. Glycosylases for oxidized bases are typically bifunctional with intrinsic AP lyase activity Glycosylases not essential in mammals while APE1 is Some ends require cleaning by one of a variety of enzymes to be compatible for closing Single Nucleotide (SN) BER involves simple insertion and ligation Long Patch BER utilizes enzymes involved in DNA replication –Typically 2-8 nucleotides are added

21. Single strand break sensors: PARP1 and XRCC1

22. The major mechanisms of repair for nucleotide damage in mammals are via excision Base Excision Repair (BER) –Repair of oxidizes, alkylated or inappropriate bases as well as abasic sites –Responsible for repair of the majority of DNA damage Nucleotide Excision Repair (NER) –Mechanism of DNA excision and repair synthesis that corrects damage caused by agents that create bulky DNA adducts (e.g. thymine dimers) –Most versatile of excision repair mechanisms Mismatch Repair (MMR) –Repairs base-base mismatches and insertion/deletion mispairings arising during DNA replication and recombination

23. NER in mammals is defined by it’s relationship with XP Seven complementation groups for XP correspond to most of the factors involved in NER in mammals

24. Actually, there are two distinct pathways of mammalian NER Global genome repair (GG-NER) Transcription-coupled repair (TC-NER) These pathways differ primarily in their damage recognition Hanawalt (~1985) -> NER of mammalian genes is faster if the genes are actively transcribed; faster than repair of silent DNA, and attributable to increased rate of repair of transcribed strand of transcribed genes.

25. What does NER recognize? Both adduct and helical distortion required.

26. NER factors are freely diffusible in vivo No pre-assembled “repairosome” in extracts from yeast. Guzder et al. JBC 271:8903 (1996). Volker et al. Mol. Cell 8:213 (2001)

27. GG-NER is triggered by recognition of DNA distortion and bulky adducts Damage recognized by XPC- RAD23B complex or UV-DDB complex (not shown) Binding may trigger extra- helical flipping of damaged base Note the ultimate sites of incision shown by arrowheads Recognition has both damage and strand specificity

28. TC-NER is triggered by RNA polII stalling –RNApolII interacts with CSB during elongation –This interaction is stabilized by encounter with lesion –Note CSB—Cockayne Syndrome B –CSB is essential for recruitment of other factors –Assembly of complex triggers chromatin remodeling –Repair may require “backtracking of RNApolII and cleavage of protruding mRNA for transcription restart

29. Incision and Repair Lesion demarcation Lesion is verified by localized unwinding and RPA binding Dual incision Damaged strand is incised at single to double strand boundaries (25-30 nts) Gap filling and ligation Different combinations of polymerases and ligases in dividing and non-dividing cells

30. Xeroderma pigmentosum Severe predisposition to skin cancers (squamous and basal cell carcinomas) Cells from patients have defects in NER Analysis of heterokaryons (cell fusions) -> 7 complementation groups, XP-A -> XP-G (plus XPV -> TLS polymerase—more on this later) Mice with XP mutations have similar phenotypes -> See other kinds of cancers not picked up because XP in humans is so rare. Cancer risk in heterozygous humans? More on this later, too

31. Cockayne syndrome A conundrum: often see photosensitivity-> repair defect but no predisposition to skin cancer ‘pure’ CS (no XP features)-> defects in TCR only CS due to defects in CSA or CSB -> TCR. CSA -> E3 ligase subunit targeting XPC CSB -> Snf2/Swi2 ATPase: nucleosome remodeling? ejection of stalled RNA polymerase? Functions of CSA and CSB obviously different but syndromes generally indistinguishable

32. Requirement for NER factors for mammalian development ERCC1 mutant mice are inviable, and no humans with mutations in ERCC1 have been identified -> critical role for ERCC1 in development some XP/CS individuals with defects in XPB, XPD (TFIIH) or XPG (endonuclease) have increased cancer risk, some do not Trichothiodystrophy (TTD): mutations in XPB or XPD sensitivity to sunlight; many have defects in NER + developmental problems Allele-specific defects in TFIIH can potentially explain diverse phenotypes NER-specific defects in TFIIH -> ‘typical’ XP NER and transcription defects of TFIIH -> XP/CS transcription-specific defects in TFIIH -> TTD

33. Repair vs Transcriptional Roles for XP/CS Proteins Cleaver Nature Rev. Cancer 5:564 (2005)