DNA  DNA Replication

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DNA DNA Replication

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1. Chapter 5 & 6 DNA & DNA Replication

2. History DNA Comprised of genes In non-dividing cell nucleus as chromatin Protein/DNA complex Chromosomes form during cell division Duplicate to yield a full set in daughter cell

3. DNA is Genetic Material

4. From Chapter 2 Nucleic acids are polymers Monomers are called nucleotides Nucleotides = base + sugar + phosphate Base = purine or pyrimidine Purines = adenine, guanine Pyrimidines = thymine, cytosine, uracil Sugar = deoxyribose or ribose Phosphate, a single phosphate in DNA Sugar of nt 1 is linked to the phosphate of nt 2 by a phosphodiester bond

5. Panel 2-6

6. Chapter 2 contd

7. DNA is a Double Helix Nucleotides A, G, T, C Sugar and phosphate form the backbone Bases lie between the backbone Held together by H-bonds between the bases A-T 2 H bonds G-C 3 H bonds

8. H - Bonds Base-pairing rules A?T only (A?U if DNA-RNA hybrid) G?C only DNA strand has directionality one end is different from the other end 2 strands are anti-parallel, run in opposite directions Complementarity results Important to replication

9. Helical Structure

10. Nucleotides as Language We must start to think of the nucleotides A, G, C and T as part of a special language the language of genes that we will see translated to the language of amino acids in proteins

11. Genes as Information Transfer A gene is the sequence of nucleotides within a portion of DNA that codes for a peptide or a functional RNA Sum of all genes = genome

12. DNA Replication Semiconservative Daughter DNA is a double helix with 1 parent strand and 1 new strand Found that 1 strand serves as the template for new strand

13. DNA Template Each strand of the parent DNA is used as a template to make the new daughter strand DNA replication makes 2 new complete double helices each with 1 old and 1 new strand

14. Replication Origin Site where replication begins 1 in E. coli 1,000s in human Strands are separated to allow replication machinery contact with the DNA Many A-T base pairs because easier to break 2 H-bonds that 3 H-bonds Note anti-parallel chains

15. Replication Fork Bidirectional movement of the DNA replication machinery

16. DNA Polymerase An enzyme that catalyzes the addition of a nucleotide to the growing DNA chain Nucleotide enters as a nucleotide tri-PO4 3OH of sugar attacks first phosphate of tri-PO4 bond on the 5 C of the new nucleotide releasing pyrophosphate (PPi) + energy

17. DNA Polymerase Bidirectional synthesis of the DNA double helix Corrects mistaken base pairings Requires an established polymer (small RNA primer) before addition of more nucleotides Other proteins and enzymes necessary

18. How is DNA Synthesized? Original theory Begin adding nucleotides at origin Add subsequent bases following pairing rules Expect both strands to be synthesized simultaneously This is NOT how it is accomplished

19. Why DNA Isnt Synthesized 3?5

20. How is DNA Synthesized? Actually how DNA is synthesized Simple addition of nucleotides along one strand, as expected Called the leading strand DNA polymerase reads 3 ? 5 along the leading strand from the RNA primer Synthesis proceeds 5 ? 3 with respect to the new daughter strand Remember how the nucleotides are added!!!!! 5 ? 3

21. How is DNA Synthesized? Actually how DNA is synthesized Other daughter strand is also synthesized 5?3 because that is only way that DNA can be assembled However the template is also being read 5?3 Compensate for this by feeding the DNA strand through the polymerase, and primers and make many short segments that are later joined (ligated) together Called the lagging strand

22. DNA Replication Fork Fig 6-12

23. Mistakes during Replication Base pairing rules must be maintained Mistake = genome mutation, may have consequence on daughter cells Only correct pairings fit in the polymerase active site If wrong nucleotide is included Polymerase uses its proofreading ability to cleave the phosphodiester bond of improper nucleotide Activity 3 ? 5 And then adds correct nucleotide and proceeds down the chain again in the 5 ? 3 direction

24. Proofreading

25. Starting Synthesis DNA polymerase can only ADD nucleotides to a growing polymer Another enzyme, primase, synthesizes a short RNA chain called a primer DNA/RNA hybrid for this short stretch Base pairing rules followed (BUT A-U) Later removed, replaced by DNA and the backbone is sealed (ligated)

26. Primers contd Simple addition of primer along leading strand RNA primer synthesized 5 ? 3, then polymerization with DNA Many primers are needed along the lagging strand 1 primer per small fragment of new DNA made along the lagging strand Called Okazaki fragments

27. Removal of Primers Other enzymes needed to excise (remove) the primers Nuclease removes the RNA primer nucleotide by nucleotide Repair polymerase replaces RNA with DNA DNA ligase seals the sugar-phosphate backbone by creating phosphodiester bond Requires Mg2+ and ATP

28. Other Necessary Proteins Helicase opens double helix and helps it uncoil Single-strand binding proteins (SSBP) keep strands separated large amount of this protein required Sliding clamp Subunit of polymerase Helps polymerase slide along strand All are coordinated with one another to produce the growing DNA strand (protein machine)

29. Components of the DNA Replication

30. Polymerase & Proteins Coordinated One polymerase complex apparently synthesizes leading/lagging strands simultaneously Even more complicated in eukaryotes

31. DNA Repair For the rare mutations occurring during replication that isnt caught by DNA polymerase proofreading For mutations occurring with daily assault If no repair In germ (sex) cells ? inherited diseases In somatic (regular) cells ? cancer

32. Effect of Mutation

33. Uncorrected Replication Errors Mismatch repair Enzyme complex recognizes mistake and excises newly-synthesized strand and fills in the correct pairing

34. Mismatch Repair contd Eukaryotes label the daughter strand with nicks to recognize the new strand Separates new from old

35. Depurination or Deamination Depurination removal of a purine base from the DNA strand Deamination is the removal of an amine group on Cytosine to yield Uracil Could lead to the insertion of Adenine rather than Guanosine on next round

36. Chemical Modifications

37. Thymine Dimers Caused by exposure to UV light 2 adjacent thymine residues become covalently linked

38. Repair Mechanisms Different enzymes recognize, excise different mistakes DNA polymerase synthesizes proper strand DNA ligase joins new fragment with the polymer

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