1. BM102 Molecular Bioscience MUTAGENESIS AND REPAIR Dr Ellis

2. Objectives By the end of this lecture you should be able to: -Appreciate the ways in which mutations can arise both spontaneously and due to chemicals or radiation damage -Understand the role of DNA repair

3. Mutation Mutate means to change DNA mutation means a change in the nucleotide sequence This can be a change to a nucleotide or a break or rearrangement in the DNA Factors that cause changes in DNA are called mutagens Mutation creates variation

4. Purine A Purine G Pyrimidine T Pyrimidine C Reminder: nucleotides and their bases

5. Mutations at the level of DNA sequence Point mutations - replace one nucleotide with an other Described in terms of the change in the nucleotide’s base: Transition purine changed for purine (A G) pyrimidine for pyrimidine (T C) Transversion purine changed for pyrimidine (G C) pyrimidine for purine (A T)

6. How point mutations happen Mutations can be Spontaneous or Chemical-induced by a mutagen that causes Changes to a base or entire nucleotide - deamination - depurination –Base analogues –Intercalating agents –UV-irradiation

7. Deamination of bases Cytosine C can spontaneously lose an amino group (NH 2 ) to form uracil U In DNA, C base pairs with G But a U would base pair with A, hence A G-C pair is converted to an A-T pair A::T | C::G | T::A A::T | U::A | T::A

8. Deamination can also be caused by reactive chemicals Nitrous acid deaminates the base to a keto group But hypoxanthine then base pairs with cytosine Thus an A-T pair has been converted into a G- C pair C::G | A::T | T::A C::G | H::C | T::A C::G | G::C | T::A AdenineHypoxanthine

9. Depurination of nucleotides The entire purine base is detached from the sugar Without any base to pair with, incorrect nucleotides are inserted during DNA replication Heat can cause spontaneous depurination – about 10,000 sites every day in a human cell!!

10. Depurination can also be caused by reactive chemicals Some reactive chemicals can cause alkylation (methylation) of bases. Ie the addition of a methyl (CH 3 ) group. Alkylation of guanine makes the bond between the base and the sugar more sensitive to breakage ie alkylation leads to depurination

11. Alkylating agents: Reactive chemicals that can alkylate DNA: Cause serious damage to cells and to DNA Chemical warfare

12. Alkylating agents can also cause mispairing: Ethyl methanesulphonate alkylates guanine to O-6-ethylguanine O-6-ethylguanine base pairs with T so changes G-C pair to A-T pair

13. Base analogues Mimic bases and incorporates into DNA in place of them Eg 5-bromouracil is an analogue of thymine

14. 5-bromouracil Exists in two forms – keto or enol Each form has a different H-bonding partner In the keto form it base pairs with A and in the enol form it base pairs with G Converts A-T pair to G-C pair Or G-C pair to A-T pair

15. Intercalating agents Planar molecules that fit in between the bases Eg acridine, proflavin, ethidium bromide Insert by intercalation Moves the bases apart, so that mistakes are made during DNA replication Addition/deletion of single bases – causes frameshift mutations

16. UV irradiation Ultraviolet (UV) light is electromagnetic radiation with a shorter wavelength than visible light, longer than X-rays. Causes dimerization of adjacent pyrimidines (mainly thymines) T pairs with next door T, instead of across the helix This distorts the helix, making H- bonding impossible Blockage in DNA replication C::G | \ A T | :: | A T | / C::G | A::T | A::T | C::G

17. Chromosomal Alterations Caused by: 1.Ionizing radiation 2.Mobile genetic elements

18. Ionising Radiation (Radioactivity) X-rays,  -rays,  and  particles High energy particles or waves Released from radioisotopes as they decay

19. Direct damage X-rays,  and  -particles Cause bond breaks on the sugar- phosphate backbone   Bond breaks

20. Indirect damage These free radicals cause the damage to DNA e-e- X ray  ray P+P+ O H H OH - H+H+ HoHo OH o X-rays and  -rays react with water to produce highly reactive ions known as free radicals (unpaired electrons)

21. Types of Damage caused by Ionizing Radiation 1)Alterations in nucleotides 2)Single stranded breaks, in the sugar- phosphate backbone 3)Double stranded breaks Attempts to repair breaks can lead to chromosome translocations, duplications, inversions and deletions

22. There is NO minimum dose Data from Drosophila The frequency of mutation is proportional to the dose of the radiation Very low doses of radiation still cause mutation – no lower thresholds. X-rays can alter the frequency of mutations

23. All mutagens increase mutation rate Chemicals and radiation that cause changes to DNA increase the mutation rate This can be measured using specific assays –Ames Test – uses bacteria –Mammalian cell culture tests All new chemicals, drugs, have to go through these test

24. DNA damage can be repaired Cell has repair systems for DNA that can correct changes before they become fixed in the genome as mutations. Enzymes can remove the incorrect base and/or nucleotide and allow the correct nucleotide to be incorporated Base excision repair Nucleotide excision repair Mismatch repair

25. Base excision repair (BER) Repairs damage to a single base Damaged base is removed by a DNA glycosylase. The sugar-phosphate then removed by AP endonuclease New nucleotides inserted by DNA polymerase, Joined up by DNA ligase

26. Nucleotide excision repair (NER) Removes pyrimidine dimers. A complex formed by 3 proteins removes the whole region of nucleotides DNA polymerase then fills in the gap and its joined by DNA ligase

27. Mismatch repair (MMR) Corrects errors of DNA replication. A protein called MutS scans the new strand and looks for mismatches It then recruits MutL

28. Mismatch repair…. And an endoclease that cuts the DNA upstream The DNA is unwound The protein RecJ removes the error strand A new strand is synthesized by DNA polymerase and joined by DNA ligase

29. Photoreactivation Enzymes called Photolyases can repair damage caused by UV light -Repairs pyrimidine dimers (no excision) -Blue light is required to activate the photolyase

30. Animation http://highered.mcgraw- hill.com/sites/dl/free/0072556781/192785/an im0035.swf http://highered.mcgraw- hill.com/sites/dl/free/0072556781/192785/an im0035.swf

31. Repairing Double strand breaks in DNA Very difficult to do but three mechanisms: 1.Homologous recombination 2.Non-homologous end joining (NHEJ) A specialized DNA ligase is involved 3.Microhomology-mediated end joining (MMEJ),

32. Homologous recombination Double and single strand breaks trigger recombination between homologous chromosomes

33. RecBCD initiates homologous recombination in bacteria at Chi sites In bacteria the RecBCD enzyme initiates homologous recombination Chi sites have a special DNA sequence that RecBCD binds to These sites are Recombination “hot spots”

34. Homologous recombination can repair double strand breaks BRCA1 and BRCA2 are proteins that help repair double strand breaks Mutations in BRCA1 and BRCA2 lead to increased risk of breast cancer Cells that cannot carry out homologous recombination very well are more susceptible to ionizing radiation

35. Mistakes in recombination Recombination between non-homologous chromosomes can lead to translocation Eg Philadelphia chromosome translocation between chromosomes 9 and 22 Associated with chronic myleoid leukaemia (CML) The junction causes an altered protein to be made

36. Translocation

37. Other recombination mistakes Failed attempts to repair double strand breaks by homologous recombination Can result in duplication, deletion, inversion events Leading to extra copies of genes, loss of genetic material Also can lead to non-disjunction – where chromosomes incorrectly separate at meiosis

38. Deletion

39. Inversion

40. Duplication

41. Mobile Genetic Elements “Jumping Genes” or Transposable Elements Pieces of DNA that can “jump” ie move around the genome Fragments of DNA are transposed ie cut out and pasted in somewhere else in the genome = Transposition ExcisionInsertion Transposable element

42. Transposable elements Transposable elements were first discovered in maize by Barbara McClintock in 1948 for which she was awarded a Nobel Prize in 1983. Element could move within the maize genome..

43. Transposable elements are found in many genomes: In maize Ac/Ds elements In fruit flyP elements In bacteriaTransposons and IS elements Multiple copies of members of the same family of elements are found throughout the genome

44. Transposable elements can cause insertion mutations Many transposable elements are in non- essential regions of the chromosome But when they transpose into an active region, it results in an insertion. the gene is ‘knocked out’ – there is no gene product (protein) made - or a truncated gene product - a severe mutation. Protein-coding gene Protein X NO PROTEIN

45. Transposable elements lead to changes in phenotype In maize, transposable elements can lead to changes in expression of genes near insertion site. In fruit fly, half of the apparent phenotypes are due to transposable elements In mouse 10% of new mutations are from transposition. Humans are more stable! eg a change in the color of corn kernels from purple to yellow

46. Transposons can carry antibiotic resistance In bacteria, transposons often contain antibiotic resistance genes. These move not only within the bacterial genome, but also to other species of bacteria via carrier molecules of DNA known as plasmids Leads to the spread of antibiotic resistance between bacteria

47. Transposable Elements in Humans Approximately 50% of the human genome ! Most are no longer ‘mobile’ (thankfully) Used to be thought of as ‘junk’ DNA…but do they have a purpose?? Most common are retrotransposons

48. Retrotransposons Use an RNA intermediate during transposition. Non-viral retransposons: Short interspersed nuclear elements -SINE sequences eg Alu Long interspersed nuclear elements -LINE sequences eg Line1 These elements don’t duplicate the target site sequence Viral retrotransposons inlude retroviruses such as HIV

49. Summary Changes to DNA can occur spontaneously or can be induced by chemicals or radiation Mutations can be single nucleotide changes or larger chromosome breaks or insertions Damage to DNA can be repaired by the cell