Dna damage


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Dna damage

  2. 2. DNA Damage  The vast majority of DNA damage affects the primary structure of the double helix  Occurs at a rate of 1,000 to 1,000,000 molecular lesions per cell per day, only 0.000165% of the human genome's approximately 6 billion bases (3 billion base pairs)
  3. 3. Damage from where? • Consequences of DNA replication errors • Chemical agents acting on the DNA • UV light imparting energy into DNA molecule • Spontaneous changes to the DNA
  4. 4. Fate of DNA damage • Tolerated (ignored) • Repaired • Can kill the cell or cause the cell to kill itself • Can become fixed, resulting in a mutation (Note: fixed = NOT repaired)
  5. 5. Examples of mutation fixation • Replication of an unrepaired misincorporation • Replication of an unrepaired cytosine deamination (deaminated cytosine = uracil)
  6. 6. Human Genome • Haploid size = 3300 Megabase pairs = 3.3 x 109 (= 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
  7. 7. How big is the problem? • Consider this: 10 bp is one helical turn which is 0.34nm (3.4x10-10 m) There are 3X109 bp of DNA per haploid human genome There are 2 genomes/cell (diploid) There are approximately 1014 cells/individual So: (3.4x10-10) (3X109) (2) (1014) = 2 x 1014 meters 2 x 1014 meters/ 3 x 108 m/sec = 6.7 x 105 light sec 6.7 x 105 /60sec/60min/24hr = 7.7 light days
  8. 8. Common types of DNA damage -- 1 1. 2. Depurination : A, G Deamination : C --> U, A --> Hypoxanthine
  9. 9. Common types of DNA damage Pyrimidine dimers (UV induced).
  10. 10. DNA repair • Damaged DNA must be repaired • If the damage is passed on to subsequent generations, then we use the evolutionary term - mutation. It must take place in the germ cells - the gametes - eggs and sperm • If damage is to somatic cells (all other cells of the body bar germ cells) then just that one individual is affected.
  11. 11. Why repair DNA? • DNA pol does a great job, but not good enough • Introduces errors in about 1 in 10E7 nucleotides added, which it does not correct • Other mechanisms exist (as we will see) to correct many of the errors left by the replication system • Most mistakes and damage corrected (99% leaving just a few - only 1 in 10E9 errors are left) • Mutations are permanent changes left in the DNA
  12. 12. Diverse DNA repair systems General mechanisms shared in eukaryotes • • • • • 1. Direct repair e.g. pyrimidine dimers 2. Base excision repair 3. Nucleotide excision repair 4. Mismatch repair 5. Recombination repair 6. SOS response (SOS)
  13. 13. • Two thymines connected together by UV light. • Photoreactivation (bacteria, yeast, some vertebrates not humans) DNA photolyase
  14. 14. Base excision repair Damaged base Base excision repair pathway (BER). (a) A DNA glycosylase recognizes a damaged base and cleaves between the base and deoxyribose in the backbone. (b) An AP endonuclease cleaves the phosphodiester backbone near the AP site. (c) DNA polymerase I initiates repair synthesis from the free 3’ OH at the nick, removing a portion of the damaged strand (with its 5’3’ exonuclease activity) and replacing it with undamaged DNA. (d) The nick remaining after DNA polymerase I has dissociated is sealed by DNA ligase. AP= apurinic or apyrimidinic (a=without)
  15. 15. A DNA glycosylase initiates base excision repair Damaged base Examples of bases cleaved by DNA glycosylases: Uracil (deamination of C) 8-oxoG paired with C (oxidation of G) Adenine across from 8-oxoG (misincorporation) Thymine across from G (5-meC deamination) Alkyl-adenine (3-meA, 7-meG, hypoxanthine)
  16. 16. Nucleotide excision repair UvrA recognizes bulky lesions UvrB and UvrC make cuts Structural distortion = signal UvrD (a) Two excinucleases (excision endonucleases) bind DNA at the site of bulky lesion. (b) One cleaves the 5’ side and the other cleaves the 3’ side of the lesion, and the DNA segment is removed by a helicase. (c) DNA polymerase fills in the gap and (d) DNA ligase seals the nick.
  17. 17. Two pathways of increasing complexity Base Excision repair Nucleotide Excision repair
  18. 18. Mismatch repair Which strand is new and which is the parent? Mut S binds mismatch Mut L links S to H Mut H recognizes the parental strand
  19. 19. Mismatch repair -- Recognition Which strand is new and which is the parent? The mutation is in the new strand! A-CH3 marks the parental strand! MutS - Binds mismatch MutL - links MutH and MutS MutH - Binds GmeATC DNA is threaded through the MutS/MutL complex. The complex moves simultaneously in both directions along the DNA until it encounters a MutH protein bound at a hemimethylated GATC sequence. MutH cleaves the unmethylated strand on the 5’ side of the G in the GATC sequence.
  20. 20. Mismatch repair -- Resolution 1. The combined action of DNA helicase II, SSB, and one of many different exonucleases (only two are labeled) removes a segment of the new strand between the MutH cleavage site and a point just beyond the mismatch. 2. The resulting gap is filled in by DNA polymerase III, and the nick is sealed by DNA ligase.
  21. 21. Thymine dimer 3’ 5’ Recombinational 5’ 3’ 3’ 5’ Thymine dimer Repair The gap in the undamaged parental strand is filled by DNA pol I and ligase. The thymine dimer can now be repaired by excision repair 3’ 5’ Thymine dimer 5’ 5’ 3’ 3’ 5’ 3’ 3’ 5’ 3’ 5’
  22. 22. SOS response • The SOS response is a global response to DNA damage in which the cell cycle is arrested and DNA repair and mutagenesis are induced. • The SOS uses the RecA protein (Rad51 in eukaryotes). • During normal growth, the SOS genes are negatively regulated by LexA repressor protein dimers . • Activation of the SOS genes occurs after DNA damage by the accumulation of ssDNA regions generated at replication forks, where DNA polymerase is blocked.
  23. 23. The SOS response • In response to extensive genetic damage there is a regulatory system that co-ordinates the bacterial cell response. This results in the increased expression of >30 genes, involved in DNA repair, these include: • recA - activator of SOS response, recombination • sfiA (sulA) - a cell division inhibitor (repair before replication) • umuC, D - an error prone bypass of thymine dimers • (loss of fidelity in DNA replication) • uvrA,B,C,D - excision repair • The SOS response is regulated by two key genes: • recA & lexA
  24. 24. Gene mutation • Mutations are heritable permanent changes in a genomic sequence. • Mutation can be caused by – spontaneous errors in DNA replication or meiotic recombination; – radiation, viruses, transposons and mutagenic chemicals; – by the organism itself, by cellular processes such as hypermutation.
  25. 25. By impact on protein sequence • • • • A frameshift mutation A nonsense mutation A Missense mutations A neutral mutation
  26. 26. A frameshift mutation • A frameshift mutation is a mutation caused by insertion or deletion of a number of nucleotides that is not evenly divisible by three from a DNA sequence
  27. 27. Mutations: Insertion A frame shift mutation Normal gene GGTCTCCTCACGCCA ↓ CCAGAGGAGUGCGGU Codons ↓ Pro-Glu-Glu-Cys-Gly Amino acids Addition mutation GGTGCTCCTCACGCCA ↓ CCACGAGGAGUGCGGU ↓ Pro-Arg-Gly-Val-Arg
  28. 28. Mutations: Deletions A frame shift mutation Normal gene GGTCTCCTCACGCCA ↓ CCAGAGGAGUGCGGU Codons ↓ Pro-Glu-Glu-Cys-Gly Amino acids Deletion mutation GGTC/CCTCACGCCA ↓ CCAGGGAGUGCGGU ↓ Pro-Gly-Ser-Ala-Val
  29. 29. A nonsense mutation • A nonsense mutation is a point mutation in a sequence of DNA that results in a premature stop codon, or a nonsense codon in the transcribed mRNA DNA: 5' - ATG ACT CAC CGA GCG CGA AGC TGA - 3'shut 3' - TAC TGA GTG GCT CGC GCT TCG ACT –5' mRNA: 5' - AUG ACU CAC CGA GCG CGA AGC UGA - 3‘ Met Thr His Arg Ala Arg Ser Stop DNA: 5' - ATG ACT CAC TGA GCG CGA AGC TGA - 3'shut 3' - TAC TGA GTG ACT CGC GCT TCG ACT –5' mRNA: Protein: Protein: 5' - AUG ACU CAC TGA GCG CGA AGC UGA - 3' Met Thr His Stop
  30. 30. A Missense mutations • A missense mutation is a point mutation in which a single nucleotide is changed, resulting in a codon that codes for a different amino acid (mutations that change an amino acid to a stop codon are considered nonsense mutations, rather than missense mutations).
  31. 31. A Missense mutations Normal gene GGTCTCCTCACGCCA ↓ CCAGAGGAGUGCGGU Codons ↓ Pro-Glu-Glu-Cys-Gly Amino acids Substitution mutation GGTCACCTCACGCCA ↓ CCAGUGGAGUGCGGU ↓ Pro-Arg-Glu-Cys-Gly
  32. 32. A neutral mutation • A neutral mutation is a mutation that has no effect on fitness. • Many or even most mutations to noncoding DNA are neutral.
  33. 33. Point mutation: a single base change CATTCACCTGTACCA GTAAGTGGACATGGT transition (T-A to C-G) normal sequence transversion (T-A to G-C) CATCCACCTGTACCA GTAGGTGGACATGGT CATGCACCTGTACCA GTACGTGGACATGGT base pair substitutions transition: pyrimidine to pyrimidine, purine to purine transversion: pyrimidine to purine
  34. 34. Deletion/ insertion CATTCACCTGTACCA GTAAGTGGACATGGT deletion CATCACCTGTACCA GTAGTGGACATGGT normal sequence insertion CATGTCACCTGTACC GTACAGTGGACATGG deletions and insertions can involve one or more base pairs
  36. 36. The consequence of mutation • Have no effect • Alter the product of a gene • Prevent the gene from functioning properly or completely • About 70 percent of these mutations having damaging effects
  37. 37. 06_19_sickle_cell.jpg
  38. 38. Evolution acts on mutations • If we did not have mutation then we would all be the same! • Any changes in the environment would be deleterious to all members of the population equally • But mutation does exist and it is supported by comparison of related organisms…
  39. 39. DNA repair defects cause disease
  40. 40. Xeroderma Pigmentosum (XP)   First discovered in late 1800s by Moritz Kaposi Severe lesions, tumors and skin deformations result from sun exposure Figures from “Living in the Shadows” article
  41. 41. Xeroderma Pigmentosum (XP) Autosomal recessive hereditary disease  Incidence: 1 in 250,000 in Western world 1 in 40,000 in Japan  Disease caused by ineffective DNA repair 
  42. 42. Xeroderma Pigmentosum (XP) Autosomal recessive hereditary disease  Incidence: 1 in 250,000 in Western world 1 in 40,000 in Japan  Disease caused by ineffective DNA repair 
  43. 43. Xeroderma Pigmentosum (XP) Median age to develop skin cancer is age 8  Incidence of skin cancer is elevated 1000 fold under the age of 20  In 1960s, proved XP was due to defective NER  Phenotype can be caused by a mutation in a number of different genes 
  44. 44. NER      XPC, XPA recognize base damage (mechanism not understood) Triggers binding of several more proteins, including XPG Binding of ERCC1-XPF endonuclease; NER complex complete Cleavage and removal of damaged oligonucleotide fragment DNA polymerase resynthesizes missing sequence and ligase reseals backbone Figure from Friedberg paper
  45. 45. What is XPF?   In the NER pathway, XPF forms a heterodimeric endonuclease with ERCC1 The ERCC1-XPF protein is responsible for splicing the 5’ end of the damaged sequence Figure from Friedberg paper
  46. 46. Treatments & Studies Prevention  Gene therapy  Dimericine (T4N5 Liposome Lotion)  Figure from Dimericine website
  47. 47. Plato and Aristotle, School of Athens. Raphael, 1508-1511. from HEREDITARY NONPOLYPOSIS COLORECTAL CANCER (HNPCC) A Review of Literature
  48. 48. Inherited colorectal carcinoma: 1. Hereditary non-polyposis colorectal cancer (57% of cases of CRC) 2. Familial adenomatous polyposis and attenuated FAP(1%) 3. Familial colorectal cancer (10-15%) 4. Peutz-Jeghers syndrome (<1%) 5. Juvenile polyposis syndrome (<1%)
  49. 49. Inherited colorectal carcinoma: 1. Hereditary non-polyposis colorectal cancer (5-7% of cases of CRC) 2. Familial adenomatous polyposis and attenuated FAP(1%) 3. Familial colorectal cancer (10-15%) 4. Peutz-Jeghers syndrome (<1%) 5. Juvenile polyposis syndrome (<1%)
  50. 50. Historical perspectives.     In 1985, the chairman of pathology at the University of Michigan, Dr. Alder Warthin, recognised this syndrome. Dr. Warthin’s seamstress prophesied that she would die of cancer because of her strong family history of endometrial, gastric and colon cancer. Dr. Warthin’s investigations of her family’s medical records revealed a pattern of autosomal dominant transmission of cancer risk. Dr. Henry Lynch fully investigated this entity in the early 1990’s and 2 hereditary syndromes were described: Lynch I and Lynch II.
  51. 51. Lynch syndromes. Lynch I  Cancer of the colon  occurring at a relatively young age (mean, 44 years),  with proximal distribution (70% of cancers located in the right colon),  predominance of mucinous or poorly differentiated (signet cell) adenocarcinoma, and the presence of tumour-infiltrating and peritumoural lymphocytes  increased number of synchronous and metachronous cancers  despite all the these poor prognostic indicators, a relatively good outcome after surgery.
  52. 52. Lynch syndromes. Lynch II  Families are at increased risk for colorectal cancer and extracolonic cancers,  endometrial  ovarian  gastric  small intestinal  pancreatic  ureteral  renal pelvic.
  53. 53.  HNPCC is inherited in an autosomal dominant fashion  HNPCC accounts for 5-7% of colorectal cancers, it is the most frequent inherited CRC syndrome in the West.  Results from a mutation in one of the DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS1, PMS2, MLH3, MSH3).
  54. 54. Genetics.   Lynch HT, de la Chapelle A: Hereditary colorectal cancer. N Engl J Med 348:919, 2003. Only 50-70% of patients meeting clinical criteria for HNPCC have an identifiable germline MMR mutation which suggests that one or more unidentifiable genes or other genetic events (e.g., large germline deletions) may be involved that result in microsatellite instability in 80-90% of cases. 20% of newly discovered cases have a spontaneous germline mutation, with no evident family history.
  55. 55. Clinical features.  Early-onset CRC.  Predominance of lesions proximal to the splenic flexure (60-70%).  Benign and malignant extracolonic tumours.  Predilection for synchronous and metachronous colorectal tumours.  The lifetime risk of CRC in HNPCC patients is approx. 80%.  A better prognosis for cancer patients with HNPCC than for nonHNPCC patients with cancer of the same stage!!!
  56. 56. Clinical features. At a mean age of 44-46 years, 78-80% of MMRpositive patients develop CRC 43% develop endometrial cancer 19% develop gastric cancer 18% develop urinary tract cancer 9% develop ovarian cancer 9% develop gallbladder and biliary cancer <5% develop central nervous system cancer <5% develop small bowel cancer ACS Surgery: Principles & Practice, 2007.
  57. 57. Investigations.  Colorectal cancer, or an HNPCC-related cancer, arising in a person < 50 years should raise the suspicion of this syndrome.  The mainstay of the diagnosis of HNPCC is a detailed family history, yet 20% of newly discovered cases are caused by spontaneous germline mutations.  CRC patients who belong to known HNPCC kindreds, who have a pedigree suggestive of HNPCC, or who meet the Bethesda criteria should be offered screening by MSI testing.  Patients with MSI-high tumours should undergo testing for germline MMR mutations. If a mutation is identified then other family members can be tested after obtaining genetic counselling. Lynch HT, de la Chapelle A: Hereditary colorectal cancer. N Engl J Med 348:919, 2003.
  58. 58. Screening and surveillance.  If a family member has tested negative for a specific MMR mutation identified in an index case => an average risk subject.  If no genetic counselling, or positive for a given MMR mutation => screening recommendation as follows: HNPCC Screening tool Recommendation CRC Colonoscopy Every 2 years beginning age 20 years, annually after 40 or 10 years younger than earliest case in family Endometrial cancer Pelvic exam, transvaginal ultrasound, endometrial aspirate, CA 125 Every 1-2 years beginning age 25-35 years Upper urinary tract cancer USS, U/A Every 1-2 years beginning at age 30-35 years Gastric cancer Upper GI endoscopy Annual after age 25-35 years CNS cancer - - Small bowel cancer - -
  59. 59. Management. Surgical therapy: When? What? Who? Prophylactic surgical intervention is considered for the following reasons: 1. 80% lifetime risk of developing CRC 2. 45% rate of metachronous tumours 3. The possibility of an accelerated adenoma-carcinoma sequence. Candidates are HNPCC patients as defined by: 1. their genotype 2. Amsterdam or Bethesda criteria 3. colon cancer and more than one advanced adenoma. Options include: 1. Prophylactic total abdominal colectomy with ileo-rectal anastomosis 2. Total proctocolectomy proctocolectomy) 3. Segmental colectomy with yearly colonoscopy. with ileal pouch-anal anastomosis (restorative
  60. 60. Management. Extracolonic disease. Endometrial and ovarian cancer.  Female patients with a family history of uterine cancer should be offered prophylactic total abdominal hysterectomy + oophorectomy (TAHBSO) if childbearing is complete or if undergoing abdominal surgery for other conditions.  43% rate of endometrial cancer in mutation-positive patients.  Inefficacy of screening for uterine cancers.  The optimal timing is unclear yet cases < 35 years have been reported.  Recommendation: begin surveillance at 25 years and delay prophylactic surgery until childbearing is complete. Lynch HT, Riley BD, Weissman SM, et al; Hereditary nonpolyposis colorectal carcinoma (HNPCC) and HNPCClike families: problems in diagnosis, surveillance, and management. Cancer 100:53, 2004.
  61. 61. Familial colorectal cancer.  Non-syndromic familial colorectal cancer accounts for 10-15% of CRC cases.  The lifetime risk of developing CRC increases with a family history of the disease and the age of onset in family.  Average risk in USA: 6%  If one first-degree relative affected: 12%  If two first-degree relatives affected: 35%  Screening colonoscopy is recommended every 5 years beginning at age 40 years or 10 years earlier than the index case.  No specific genetic abnormalities are associated with familial CRC.
  62. 62. The take-home message.  Every patient presenting with a malignant tumor involving the colon, rectum, stomach, uterus, ovaries, renal pelvis or ureters under the age of 50 years is to be screened or at least counseled for a possible inherited carcinoma syndrome.  He/she and their first-degree relatives are to be counseled appropriately with the aid of a geneticist, if possible.
  63. 63. Thank u