Genetic Material-DNA

1. Genetic Material-DNA 6 November 2003 Reading:The Cell; Chapter 5, pages: 192-201

2. DNA Repair • In the living cell, DNA undergoes frequent chemical change, especially when it is being replicated. Most of these changes are quickly repaired. • A failure to repair DNA produces a mutation • The human genome has already revealed 130 genes whose products participate in DNA repair.

3. Agents that Damage DNA • Certain wavelengths of radiation • ionizing radiation such as gamma rays and x-rays • ultraviolet rays, especially the UV-C rays (~260 nm) that are absorbed strongly by DNA but also the longer-wavelength UV-B that penetrates the ozone shield. • Highly-reactive oxygen radicals produced during normal cellular respiration as well as by other biochemical pathways.

4. Agents that Damage DNA • Chemicals in the Environment • many hydrocarbons, including some found in cigarette smoke • some plant and microbial products • Chemicals used in chemotherapy, especially chemotherapy of cancers

5. Types of DNA Damage • All four of the bases in DNA (A, T, C, G) can be covalently modified at various positions.

6. Types of DNA Damage • Spontaneous damage to DNA. • One of the most frequent is the loss of an amino group ("deamination") - resulting, for example, in a C being converted to a U.

7. Types of DNA Damage • Spontaneous damage to DNA. • Depurination: cleavage of the bond between the purine bases and the sugar, leaving apurinic site (AP) in DNA

8. Types of DNA Damage • DNA damage induced by radiation and chemicals. • Formation of pyrimidine dimers.

9. Types of DNA Damage • Alkylation: addition of methyl or ethyl groups to various positions on the DNA bases. Instead of C, T is put to complement G.

10. Types of DNA Damage • Reaction with carcinogens: many carcinogens results in the addition of bulky groups to the DNA molecule

11. What can be done to repair the damage?

12. DNA Repair • Direct reversal of of the chemical reaction that causes DNA damage • Removal of the damaged base.

13. Types of DNA Damage • DNA damage induced by radiation and chemicals. • Formation of pyrimidine dimers.

14. Direct Reversal of Base Damage • Pyrimidine dimers • UV-induced damage causes skin cancers. • Cyclobutane ring results from the saturation of the double bonds between carbons 5 and t. • Formation of such dimers distort DNA structure • Photoreactivaton provides energy to break the cyclobutane ring. Humans lack this mechanism.

15. Types of DNA Damage • Alkylation: addition of methyl or ethyl groups to various positions on the DNA bases. Instead of C, T is put to complement G.

16. Direct Reversal of Base Damage • Alkylated guanine residues results from exposure to alkylating agents. • They can transfer methyl or ethyl groups to DNA. • O6 -methylguanine transferase transfers a methyl group from DNA to a cysteine residue in its active site. Humans have this mechanism.

17. Excision Repair • General means to repair DNA. • Damaged DNA is recognized and removed as free bases or as nucleotides. • The resulting gap is filled. • Uracil is occationally incorporated in place of Tymine and should be removed. • Uracil can be formed by deamination of cytosine.

18. Base Excision Repair • Removal of the damaged base. “Base excision repair”. This is done by a DNA glycosylase. • Removal of its deoxyribose phosphate in the backbone, producing a gap. • Replacement with the correct nucleotide. This relies on DNA polymerase , • Ligation of the break in the strand with DNA ligase. This requires ATP to provide the needed energy.

19. Nucleotide excision repair • Widespread form of DNA repair. • Damaged bases are removed as part of an oligonucleotide containing the lesion. • UV induced pyrimidine dimers and bulky group addition can be repaired by this mechanism.

20. Nucleotide excision repair • The damage is recognized by one or more protein factors that assemble at the location. • Cuts are made on both the 3' side and the 5' side of the damaged area so the tract containing the damage can be removed. • DNA synthesis - using the intact (opposite) strand as a template - fills in the correct nucleotides. • A DNA ligase covalent binds the fresh piece into the backbone

21. In E.coli • Three genes, uvrA, uvrB, uvrC. • What happens if these genes are mutated? • The bacteria become highly sensitive to UV (gets damaged by it). • UvrA-recognizes the damaged DNA and recruits UvrB and UvrC to the damaged area. • UvrB and UvrC then cleave the 3’ and 5’ sides of the damaged site. • UvrABC comples is called exinuclease (excise an oligonucleotide). • Helicase is needed to remove the damaged area; gap is filled with polymerase and ligase.

22. In eukaryotes • RAD genes (radiation sensitivity) mutants have increased sensitivity to UV exposure. • Inherited diseases that result from deficiencies in ability to repair DNA damage. • Xeroderma pigmentosum (XP)-sensitive to UV, develop skin cancers. They cant carry out nucleotide excision repair. • XPA to XPG (seven repair genes) highly homologous to yeast RAD genes.

23. Mismatch Repair • Mismatch repair deals with correcting mismatches of the normal bases; that is, failures to maintain normal Watson-Crick base pairing (A.T, C.G) • Many of the mismatched bases are removed during replication by the proofreading activity of DNA polymerase. Missed ones are subject to mismatch repair!!! • Mutations in either of these genes predisposes the person to an inherited form of colon cancer. (Do not forget to read the box @ page 198.

24. How could the mismatched base be understood? GGTACGATG CCATTCTAC

25. Mismatch repair in E. coli • Scans newly replicated DNA, if found enzymes of this system can identify and repair the mismatched base from newly replicated DNA. • In E.coli, methylation indicates parental strand; Adenine residues in the sequence GATC forms 6-methyladenine. Methylation occurs after replication.

26. Mismatch repair in E.coli • MutS protein initiates repair because it recognizes the mismatch and forms a complex with two other proteins MutL and MutH. • MutH is an endonuclease that can cleave the unmethylated DNA strand. • MutL and MutS then excise the DNA between the strand break and gap is filled with Pol and ligase.

27. Mismatch Repair in E.coli

28. Mismatch Repair in mammalian cells

29. Mismatch repair in mammalian cells • The old and new strands of DNA is distinguished by a different mechanism than methylation. • Presence of single strand breaks indicate newly replicating DNA or associations between MutS and MutL homologs also indicate which strand is new.

30. Colon Cancer • Cancers of the colon and rectum (colorectal cancers). • 140,000 cancer cases per year (10% of total cancer cases). • Mostly non inherited. • Inherited cases: • Familial adenomatous polyposis (rare, 1%) • Heretidary nonpolyposis colorectal cancer (15%).

31. Molecular Basis • Mutated genes involved in cell proliferation, leading to uncontrolled growth. • Mutations occur sporadically in somatic cells. • In hereditary cases, inherited germ-line mutations predispose the individual to cancer.

32. The gene • Human homology of E.coli MutS gene involved in mismatch repair of DNA is responsible for 50% of HNPCC. • Three other genes also involved in repair may be responsible. • Defects in these genes result in high frequency of mutations in other cells.

33. Symptoms • Development of the outgrowth of small benign polyps, which eventually become malignant. • Polyps can be removed surgically. Early diagnosis is important.

34. Postreplication Repair • Recombinational repair relies on replacement of damaged DNA by recombination with an undamaged molecule. • Happens during replication.

35. Recombinational Repair • Normal replication is blocked with a TT dimer. • Downstream of the damage replication goes on. • Undamaged parental strand (which has been replicated) is then used as a template, new strand is synthesized based on this. • TT dimer later is dealth with an excision repair mechanism.

36. Double strand breaks • X-rays induce double strand breaks on the chromosomes. • Ligate the ends of the chromosomes (risky, possible errors (loss of bases at the ends). • Homologous recombination provides new templates at the site of the double strand break.

37. Error-prone repair • Reversal and excision repair systems act to correct DNA damage before replication. • Replicative DNA synthesis requires an undamaged DNA strand as a template. • What about the damage at the replciation fork, when TT dimers for example block the replication. • Cells have specialized Polymerases to replicate across a damaged site but these polymerases lead to a lot mistakes.

38. Error-prone polymerases • In E. coli Polymerase V is induced in response to UV irradiation and can synthesize a new DNA strand across from a thymine dimer. • E. coli Pol II and Pol IV are induced by DNA damage. • Characteristically error-prone DNA polymerases exhibit low fidelity (100 to 10,000 times higher than replicative polymerases; E.coli PolII and eurkaryotic epsilon). • Error prone polymerases lack 3’ 5’ proofreading activity.