Chapter 7: DNA Repair Pathways

1. Chapter 7: DNA Repair Pathways

2. We totally missed the possible role of enzymes in DNA repair…. I later came to realize that DNA is so precious that probably many distinct repair mechanisms would exist. Nowadays one could hardly discuss mutation without considering repair at the same time. Francis Crick, Nature (1974), 248:766

3. 7.1 Introduction

4. DNA damage poses a continuous threat to genomic integrity. • Cells have evolved a range of DNA repair enzymes and repair polymerases as complex as the DNA replication apparatus itself. • DNA replication, repair, and recombination share many common features.

5. 7.2 Mutations and DNA damage

6. Spontaneous mutations • Occur as a result of natural processes in cells. e.g. DNA replication errors Induced mutations • Occur as a result of interaction of DNA with an outside agent that causes DNA damage.

7. Mutations are of fundamental importance • Mutations are important as the major source of genetic variation that drives evolutionary change. • Mutations may have deleterious or (rarely) advantageous consequences to an organism or its descendents. • Mutant organisms are important tools for molecular biologists in characterizing the genes involved in cellular processes.

8. The simplest type of mutation is a nucleotide substitution. • Mutations that alter a single nucleotide are called point mutations.

9. Transitions and transversions can lead to silent, missense, or nonsense mutations • Transition mutations replace one pyrimidine base with another, or one purine base with another. • Transversion mutations replace a pyrimidine with a purine or vice versa. • In humans, the ratio of transitions to transversions is approximately 2:1

10. A transition or transversion mutation can be permanently incorporated by DNA replication.

11. Whether or not nucleotide substitutions have a phenotypic effect depends on: • Do they alter a critical nucleotide in a gene regulatory region? • Do they alter a critical nucleotide in the template for a functional RNA molecule? • Are they silent, missense, or nonsense mutations in a protein-coding gene?

12. Silent mutations • Mutations that change the nucleotide sequence without changing the amino acid sequence are called synonymous mutations or silent mutations.

13. Missense mutations • Nucleotide substitutions in protein-coding regions that do result in changed amino acids are called nonsynonymous mutations or missense mutations. • May alter the biological properties of the protein. • Sickle cell anemia is an AT→TA transversion: • Glutamic acid codon in the -globin gene replaced by a valine codon

14. Nonsense mutations • A nucleotide substitution that creates a new stop codon is called a nonsense mutation. • Causes premature chain termination during protein synthesis. • Nearly always a nonfunctional product.

15. Insertions or deletions can cause frameshift mutations • If the length of an insertion or deletion is not an exact multiple of three nucleotides, this results in a shift in the reading frame of the resulting mRNA. • Usually leads to production of a nonfunctional protein.

16. Expansion of trinucleotide repeats leads to genetic instability • Trinucleotide repeats can adopt triple helix conformations and unusual DNA secondary structures that interfere with transcription and DNA replication. • Expansion of trinucleotide repeats leads to certain genetic neurological disorders.

17. Repeat expansion can occur by two different mechanisms: • Unequal crossing over. • Slippage during DNA replication.

18. Unequal crossing over • A trinucleotide repeat in one chromosome misaligns for recombination during meiosis with a different copy of the repeat in the homologous chromosome. • Recombination increases the number of repeats on one chromosome, resulting in a duplication. • On the other chromosome, there is a deletion.

19. Slippage during DNA replication • During DNA replication the DNA melts and then reanneals incorrectly in the repeated region, resulting in re-replication of an additional repeat.

20. General classes of DNA damage • Spontaneous damage to DNA can occur through the action of water in the aqueous environment of the cell. • A mutagen is any chemical agent that causes an increase in the rate of mutation above the spontaneous background.

21. Three general classes of DNA damage • Single base changes • Structural distortion • DNA backbone damage

22. Single base changes • A single base change or “conversion” affects the DNA sequence but has only a minor effect on overall structure. • Deamination is the most frequent and important kind of hydrolytic damage. • Methylated cytosines are “hotspots” for spontaneous mutation in vertebrate DNA because deamination of 5-methylcytosine generates thymine.

23. Alkylating agents such as nitrosamines lead to the formation of O6-methylguanosine. • This modified base often mispairs with thymine. • Can result in a GC→GT→AT point mutation after DNA replication.

24. Oxidizing agents generated by ionizing radiation and chemicals that generate free radicals can lead to formation of 8-oxoguanine (oxoG) • OxoG can form a Hoogsteen base pair with adenine. • Gives rise to a GC→TA transversion. • One of the most common mutations found in human cancers.

25. Structural distortion • UV radiation induces that formation of a cyclobutane ring between adjacent thymines, forming a T-T dimer. • The T-T dimer distorts the double helix and can block transcription and replication. • UV radiation can also induce dimers between cytosine and thymine.

26. Other bulky adducts can be induced by chemical mutagenesis. • Structural distortion can be caused by intercalating agents and base analogs: • Ethidium bromide has several flat polycyclic rings that insert between the DNA bases. • 5-bromouracil, an analog of thymine, can mispair with guanine.

27. DNA backbone damage Formation of abasic sites • Loss of the nitrogenous base from a nucleotide. • Generated spontaneously by the formation of unstable base adducts. Double-stranded DNA breaks • Induced by ionizing radiation and a wide range of chemical compounds. • The most severe type of DNA damage.

28. Cellular responses to DNA damage • Damage bypass • Damage reversal • Damage removal

29. 7.3 Lesion bypass

30. Translesion synthesis (TLS) • Specialized low-fidelity, “error-prone” DNA polymerases transiently replace the replicative polymerases and copy past damaged DNA. • Typical error rates range from 10-1 to 10-3 per base pair.

31. Error-prone DNA polymerases • May insert incorrect nucleotides opposite the lesion: nucleotide substitution • May skip past and insert correct nucleotides opposite bases downstream: frameshift • A trade-off between death and a risk of high mutation rate.

32. DNA polymerase eta () • Performs translesion synthesis past TT dimers by inserting AA. • Has an extra wide active site that can accommodate two dNTPs instead of one. • Van der Waals forces and hydrogen-bonding interactions hold the TT dimer so that the two thymines can be paired with two adenines.

33. 7.4 Direct reversal of DNA damage

34. Reversal of thymine-thymine dimers by DNA photolyase • In most organisms, UV radiation damage to DNA can be directly repaired. • DNA photolyase uses energy from near UV to blue light to break the covalent bonds holding two adjacent pyrimidines together.

35. DNA photolyase has two cofactors: • A pigment that absorbs UV/blue light • Fully reduced flavin dinucleotide (FADH-) • Splitting of the TT dimer is initiated by an electron transferred from photoexcited FADH- to the TT dimer already bound to the enzyme.

36. The TT dimer is flipped out of the DNA helix and brought very close to FADH-. • An electron is transferred from FADH- and the dimer is split. • The electron is then returned to the transiently formed flavin radical in less than a nanosecond.

37. Photolyases are an ancient and efficient means of repairing UV-damaged DNA. • Placental mammals including humans, however, do not have a photoreactivation pathway.

38. Damage reversal by DNA methyltransferase • Methyltransferase catalyzes the transfer of the methyl group on O6-methylguanine to the sulfhydryl group of a cysteine residue on the enzyme.

39. Damage reversal by DNA methyltransferase • DNA methyltransferase binds the minor groove of the DNA. • The minor groove widens and the DNA bends by 15° away from the enzyme. • The O6-methylguanine flips out from the double helix into the active site. • A sulfhydryl group of a cysteine in the active site accepts the methyl group from guanine.

40. Does DNA methyltransferase fit the classic definition of an enzyme?

41. 7.5 Repair of single base changes and structural distortions by removal of DNA damage

42. Multiple dynamic protein interactions are involved in all repair processes. • Ordered hand-off of damaged DNA from one protein or protein complex to another. • DNA repair proteins are modular.

43. The repair machinery must gain access to the DNA • Upon sensing DNA damage, nucleosomes are disassembled by histone modification and chromatin remodeling. • After repair, PCNA recruits chromatin assembly factors to restore nucleosomes.

44. Pathways for repair of single base changes and structural distortion • Single base changes • Base excision repair • Mismatch repair • Structural distortion • Nucleotide excision repair

45. Base excision repair • The correction of single base changes that are due to conversion of one base to another. • Specific DNA glycosylases recognize and excise the damaged base. • How do DNA repair proteins find the rare sites of damage in a vast expanse of undamaged DNA?

46. Model for DNA damage recognition by 8-oxoguanine DNA glycosylase 1 (hOGG1) • A series of “gates” within the hOGG1 enzyme • hOGG1 first binds nonspecifically to DNA.

47. If the enzyme encounters a normal GC base pair, then: The G is transiently extruded into a G-specific pocket and returned to the double helix. • If the enzyme encounters a oxoG-C base pair, then: The oxoG is extruded into the G-specific pocket and then inserted into a lesion recognition pocket where it is excised.

48. Base excision repair pathway in mammalian cells • A DNA glycosylase recognizes and excises the damaged base. • An endonuclease cleaves the phosphodiester bond either 3′ or 5′ of the abasic site. • An endonuclease removes 1-10 nucleotides. • DNA polymerase replaces the missing nucleotides. • DNA ligase seals the gap.

49. Mismatch repair • The correction of mismatched base pairs which result from DNA polymerase errors during replication. • A large region of DNA including the mismatch is excised. • The method of strand discrimination in mammalian cells is currently unknown.

50. Hereditary nonpolyposis colorectal cancer: a defect in mismatch repair 3 to 5% of all colorectal cancers • Inherit one inactive mismatch repair allele. • Somatic loss of wild-type allele. • Defective mismatch repair mechanism. • Accumulation of mistakes during DNA replication. • Microsatellite instability. 80% lifetime risk of developing colorectal cancer.