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BIOL 308
Dragana Miskovic

1. Distinguish between the terms “mutation”, “DNA repair” and “recombination”. A mutation is an error in the DNA that’s not repaired before the next replication cycle and therefore, becomes permenant. Recombination is a process by which a strand of DNA is first denatured from its dsDNA state and then reannealed to a different ssDNA strand from the same organism or homologous gene from another organism. DNA repair are intrinsic mechanisms used by the body to repair faults in DNA and prevent mutations. 2. List and briefly explain three major causes for mutation in DNA. a) Replication errors: Mistakes made during replication that were not detected and corrected by pol I or III proofreading mechanisms. (A instead of C) if not corrected, leads to loss of homologous chromosome. (one will have AT, other CG) b) Spontaneous changes in DNA: i. 5000 depurinations/human cell/day. (base is lost. The complementary base is deleted and a frame shift occurs) ii. 100 spontaneous deaminations of C into U/human cell/day. iii. A is converted to hypoxanthine which pairs C not T. c) External factors: Radiation, changes in temperature, mutagens etc. A. Alkylating chemicals: adds alkyl group to DNA B. Reactive oxygen species: base pairs with A and C. Highly mutagenic. C. Non-ionizing radiation: (UV- 260nm) highly absorbed by bases. Leads to thymine dimers that cause Xeroderma Pigmentosum D. Ionizing radiation (X rays and gamma rays) double strand breaks. E. Base analogues: 5 bromouracil=T and replaces it. F. Intercalating agents: insert between stacked DNA bases. May cause deletion or addition of bases and therefore frame shift. 1 in 10 mistakes per genome will not be repaired/cell division. So 3 in humans. N.B: Depurination is spontaneous hydrolysis of N-glycosyl linkage leading to an abasic site in DNA. (U is read as T by replication machinery) 3. Explain how errors in DNA replication can lead to mutations. Failure to repair DNA before the next round of replication will lead to permanent change in DNA (mutation). This is because the two strands will separate and the new strands formed will carry the complementary bases to the mutated bases and thus, the mutation now doesn’t need repair and becomes permenant. 4. Distinguish between the effects of mutations on the somatic and germ cells of multicellular organism. A mutation in a somatic cell is not hereditary (not passed vertically), as somatic cells do not contribute to gametes, however, the mutation can still be detrimental and can cause cancers. A mutation in the germ cells or the germ cell precursors can lead to hereditary diseases as the mutated DNA will form the original template used to make the DNA in the other cells and will therefore, be passed on. 5. List the various types of DNA repair mechanism (we have mentioned seven). a) Proof-reading by DNA polymerase (I and III) – 3’ to 5’ exonuclease. b) Direct reversal of damage c) Base excision repair d) Nucleotide excision repair e) Mismatch repair f) Recombination repair g) Error-prone repair 6. Give the detailed description of the base excision (or nucleotide excision) repair process in bacteria. Base excision repair: (lesion specific) Base excision is lesion specific. Glycosylase removes N. AP endonuclease cuts sugarphosphaste phosphodiester bonds on both sides of damaged base. Exonuclease remove sugar and phosphate. Polymerase synthesizes replacement DNA. Ligase seals nicks. Nucleotide excision repair: non lesion specific, detect distortion, UvrA scan and binds with UvrB at the site, UvrB melts DNA while A leaves and C joins, UvrC nicks both strands (7 to 5’ and 3 to 3’). 7. Describe the mismatch repair process of bacteria (pay attention at the ways in which the daughter and parent strand are recognized by repair system). a. MutS scans DNA for distortion→ binds to mismatch b. MutL recruited by MutS-mismatch-DNA complex MutS translocates along DNA until a GATC sequence is reached • requires ATP • binding of MutS at the 2 locations creates loop in DNA c. MutSL activates MutH (recognizes loop) d. MutH (endonuclease): recognizes GATC, binds MutSL e. MutH nicks unmethylated (daughter) strand of DNA → strand is then progressively excised from GATC to mismatch in 5’ to 3’ direction (using RecJ or exonuclease III) or in 3’ to 5’ direction (using exonuclease I). A helicase (UvrD) assists. New DNA strand is synthesized by DNA polymerase III and sealed by DNA ligase The repair system recognizes the daughter strand as its not methylated yet. (Dam methylates the A of GATC, so when the dimer binds to the GATC, it recognizes its methylation status and therefore, recognizes whether the strand is a parent or daughter strand) N.B: Methylation also occurs on C of CmC(A/T)GG by Dcm. Methylation of Adenine regulates replication while that of cytosine regulates transcription) 8. Describe briefly the mechanism of direct reversal of damage in bacteria. Thymine dimers are corrected by photolyase (Light-dependent activity) that breaks covalent bonds between thymines; chemical basis still unknown - photoreactivation 9. Distinguish between the two DSB repair mechanisms we talked about in class. (Double stranded breaks) Recombination repair Nonhomologous end joining Accurate Error prone Replication fork encounters lesion in DNA Both strands are broken at the same that has not been repaired place The gap is filled using a template from a Double stranded break with no homologous single strand undamaged complementary strand available Binding of both broken strands by making blunt ends. Ku70/80 dimmers connect strands. DNA dependent protein kinase (DNA-PK) brings and phosphorylates protein Artemis. Artemis has exo-endonuclease to make the ends blunt. DNAP fill in the gap. Ligase and WRCC4 join them. 10. What is SOS repair mechanism, when is it used and why is it important? It’s an induced multigene response. It’s a highly error prone repair mechanism where polymerases insert nucleotides independent of base pairing. This introduces mutations but still enables complete chromosome replication. They induce point mutations and are therefore preferred to the other repair mechanisms that lead to frame shifts. It is used when the other mechanisms fail. This is the last repair mechanism known and if it fails, the cells commit suicide by apoptosis. 11. Describe the term “non-homologous end joining” and explain how this process results in the repair of double strand breaks in DNA molecule. NHEJ is a DSB repair mechanism where the broken ends are directly ligated without the need for a homologous template. The overhangs are removed and the ends are directly ligated. This could lead to a frame shift but the chromosome is complete none the less and can be replicated. 12. What are biological roles of DNA recombination? 1) DNA
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