1. Cellular Response to DNA Damage - Repair ENVR 430: Health Effects of Environmental Agents October 9, 2009 John R. Ridpath Rosenau 347 966-6141

2. DNA Background DNA encodes all genetic information Original assumption: blue-print for life must be fundamentally stable Physicist Erwin Schrödinger (in his monograph “What is Life”, 1944): suggested changes could occur to the “hereditary code script” It was known x-rays could break chromosomes Schrödinger said the lesions could be replaced by “ingenious crossings” with the unharmed chromosome – we now call this DNA repair mechanism homologous recombination DNA primary structure elucidated in 1953

3. Terminology Remedial Mutation – heritable change in sequence of genome Mutant – organism that carries one or more mutations Genotype – genetic information organism encodes in its genome Phenotype – ensemble of observable characteristics of an organism Mutagen – agent that leads to an increase in the frequency of occurrence of mutations Mutagenesis – process by which mutations are produced

4. DNA Damage Our genome (primary structure of DNA) is continually beset with insults caused by a myriad of agents, both endogenous and exogenous to the cell.

5. After DNA Damage, then What? AcuteLong-Term Cancer Aging Degenerative disease Mutation Cell death DNA repairHealthy Slide courtesy of Brian Pachkowski

6. Sources of DNA Damage Endogenous sources  Spontaneous hydrolysis of bond between base and sugar of backbone; 18000 purines (A & G)/cell/day lost  Deamination of cytosine to uracil; 100-500/cell/day  Oxygen radicals (ROS) react with bases; Ex: 8- oxoG, 1000-2000/cell/day  Replication errors; enough errors to be devastating  Methylating agents (Ex: SAM); react with all bases, 1200/cell/day

7. Sources of DNA Damage Exogenous sources  Ionizing radiation; radioactives, cosmic rays  Man-made chemicals react with and alter DNA structure and chemistry  UV radiation from sun; fuses adjacent bases (thymine dimers)

8. Examples of DNA Damage

9. DNA Repair DNA repair “…connote(s) cellular responses to DNA damage that result in the restoration of normal nucleotide (base) sequence and DNA structure…” * * Friedberg, et al., DNA Repair and Mutagenesis, 2 nd ed.; ASM Press; Washington, D.C., 2006; p 4.

10. DNA Repair Pathways Direct reversal Mismatch Base excision/ SSB Nucleotide excision Homologous recombination Non- homologous end joining Type of Lesion O 6 - MeGuanine, Pyrimidine dimers Mispaired bases Alkylations, oxidations, abasic sites, strand breaks Bulky or helix distorting adducts Double strand breaks, crosslinks Double strand breaks Slide courtesy of Brian Pachkowski

11. Direct Reversal of DNA Damage Repairs: pyrimidine dimers (UV), methylated bases How: enzymatic reaction – just changes it back  DNA methyltransferases: proteins that remove methyl groups from bases  Cryptochrome: human enzyme that reverses pyrimidine dimers Fidelity: Most efficient, most accurate repair – single enzyme, single step Consequence of failure:  Dimers; interference with replication and transcription  methylated bases; GC → AT transitions, heritable mutations

12. Direct Reversal of DNA Damage The proteins MGMT and ABH2 are used to directly remove methyl groups in direct reversal Wyatt and Pittman, Chem. Res. Toxicol. 2006, 19, 1580-1594

13. Mismatch Repair Repairs: improperly paired nucleotides and insertion/deletion loops during replication How  searches for signal that identifies newly synthesized strand; template strand contains methylated bases, new strand is not immediately methylated  degrades this strand past mismatch  resynthesizes the excised strand Consequences of failure: increased susceptibility to cancer, especially hereditary non-polyposis colorectal carcinoma (HNPCC)

14. Mismatch Repair GATC G G 5’ GATC G 5’ 3’ GATC G 5’ CTAG T 3’ Me 5’ 3’ 5’ 1.Enzyme complex recognizes G:T mismatch in hemimethylated DNA 2.Excises mismatched nucleotide (T) on unmethylated strand and reinserts correct nucleotide

15. Base Excision Repair When thine eye offends thee … Repairs  oxidized/reduced bases (Ex: 8-oxoG, 1000- 2000/cell/day)  alkylated bases  deaminated bases  mismatched bases (replication errors)  missing bases [apurinic, apyrimidinic (AP) sites] How: removes offending base and replaces with correct base Fidelity: excellent

16. Base Excision Repair Consequences of failure  Base substitution → transitions, transversions → point mutations  AP sites  Single strand breaks that may lead to double strand breaks

17. Base Excision Repair Adapted from Sancar, et al., Annu. Rev. Biochem. 2004. 73:39-85 Short patch Long patch

18. Nucleotide Excision Repair Repairs: cyclobutane pyrimidine dimers (CPD), bulky adducts (i.e., B[a]P), AP sites, intercalated compounds, DNA interstrand crosslinks How  Recognition and verification of base damage  Incision of DNA strand on either side of damage  Excision of oligonucleotide fragment generated by incisions  Repair synthesis to fill the gap  Ligation of nick in DNA

19. Nucleotide Excision Repair Fidelity: Excellent Consequences of failure  Interference with replication, transcription

20. Nucleotide Excision Repair Adapted from Sancar, et al., Annu. Rev. Biochem. 2004. 73:39-85 Recognition and verification of damage

21. Nucleotide Excision Repair Recognition and verification of damage

22. Nucleotide Excision Repair Incision on either flank of affected strand

23. Nucleotide Excision Repair PIC 4 Excision of affected oligonucleotide and resynthesis of strand

24. Nucleotide Excision Repair PIC 5 Ligation of nick in DNA strand by DNA ligase I (not specifically shown)

25. Double Strand Break Repair Two types of DSB repair  Homologous recombination (HR)  Non-homologous end joining (NHEJ) DSB Caused by: ionizing radiation/ROS, replication fork encountering single-strand break, other repair mechanisms Experimental evidence suggests NHEJ is the primary mechanism used early in the cell cycle (G1) while HR is used later (S/G2)

26. Double Strand Break Repair Consequences of failure  Sister chromatid exchanges (SCE)  Aneuploidy – loss or duplication of chromosomes or chromosomal segments (proposed as the initiating event for cancer)

27. Double Strand Break Repair Homologous Recombination Repairs: DNA double-strand breaks How  Utilizes another DNA molecule that has a similar (homologous) or identical DNA sequence (sister chromatid)  One strand on each side of the break in the damaged molecule is degraded to leave 3’ single strands  One of the single strands then invades the homologous nucleotide sequence of the other DNA molecule using it as a template to reconstruct the damaged molecule Fidelity: Virtually error free, especially if sister chromatid is used

28. Double-strand Break Repair by Homologous Recombination Slide courtesy of Jeff Sekelsky Damage removal, resection strand invasion X X DSB Displaced yellow strand is captured by blue strand Homologous DNA strand Crossovers (Holliday junctions) are then resolved

29. Double Strand Break Repair Non-homologous end joining Double strand break repair the easy way – just deal with it How  Protect and trim the ragged ends  Bridge the gap  Ligate the nicks Fidelity: poor – deletions can result in loss of coding information

30. Non-homologous End Joining Adapted from Sancar, et al., Annu. Rev. Biochem. 2004. 73:39-85

31. Examples of Human Genetic Diseases Caused by Dysfunctional Repair Pathways Human disease Gene(s)Defective pathway Clinical features Xeroderma pigmentosum (XP) XPA-XPG; XPVNERDermatitis, skin cancer, neurological defects Nijmegen breakage syndrome (NBS) NBS1Strand break repair Developmental abnormalities growth retardation, cancer predisposition CancerBRCA1,BRCA2HRHereditary breast, ovarian cancer Fanconi anemiaFANCs,BRCA2HR Limb defects, anemia, cancer Hereditary non- polyposis colon cancer (HNPCC) MSH2, MSH3, MSH6, others Mismatch repair Colon and other cancers

32. Single Nucleotide Polymorphisms (SNP) SNP: a change in a single nucleotide on one allele when a gene on both alleles is compared Occurrence in human genome: approximately one in every ~1330 bases An allele is defined as polymorphic if it appears in > 1% of the population Can alter protein function including that of repair proteins (Ex: XRCC1 used in BER) DNA strand 1 differs from DNA strand 2 at a single base-pair location (a C/T polymorphism).

33. Mutator Phenotype Most cancer cells exhibit greater numbers of mutations than would be expected randomly Mutator phenotype: results from mutations in genes that are responsible for genomic stability (i.e., genes for repair proteins, genes responsible for the proper segregation of chromosomes during mitosis) Allows for accumulation of massive numbers of mutations Can have a cascade effect if even more repair proteins become mutated