Cellular Response to DNA Damage - Repair

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? Acute Long-Term DNA repair Healthy Cancer Mutation Aging Cell death Degenerative disease 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, 2nd ed.; ASM Press; Washington, D.C., 2006; p 4.

10. DNA Repair Pathways 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. Me Mismatch Repair 5’ 5’ 5’ 3’ G G G G T GATC GATC GATC GATC CTAG 3’ 5’ 5’ 3’ • Enzyme complex recognizes G:T mismatch in hemimethylated DNA • 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 Short patch Long patch Adapted from Sancar, et al., Annu. Rev. Biochem. 2004. 73:39-85

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

21. Nucleotide Excision Repair Recognition and verification of damage

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

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

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

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

29. Double Strand Break RepairNon-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

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