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Chapter 5: DNA Replication, Repair, and Recombination

Chapter 5: DNA Replication, Repair, and Recombination . Goals . Illustrate how structure of DNA affects its function Describe enzymes involved in replication Summarize their functions Explain how DNA is repaired and why it needs to be repaired. DNA Structure (A Review).

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Chapter 5: DNA Replication, Repair, and Recombination

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  1. Chapter 5: DNA Replication, Repair, and Recombination

  2. Goals • Illustrate how structure of DNA affects its function • Describe enzymes involved in replication • Summarize their functions • Explain how DNA is repaired and why it needs to be repaired

  3. DNA Structure (A Review) • DNA consists of two strands • Each strand is a polymer of nucleotides • Strand has orientation due to nucleotide structure: 5’ and 3’ ends • The two strands are antiparallel

  4. DNA Function (A Review) • DNA function is information storage • Sequence of strand stores info • Genes are copied into RNA (transcription) • “Control elements” regulate protein interactions with DNA • DNA passed on to descendant cells • Accurate copying • Repair of any damage to avoid changes • Accurate subdivision

  5. Functions Determine Form • Double strands of DNA allow “easy” replication • Rules for obligate pairing • Each strand acts as template for the other • Multiple proteins involved • Act in concert • Act as complexes • Recognize DNA by shape of bases

  6. Paired DNA Strands

  7. Replication Overview • Replication complex binds to replication “origin” • Double-stranded DNA is “melted” • Each strand is used as a template for DNA synthesis

  8. Mechanism of DNA Replication General Features of DNA Replication • Semiconservative • Complementary Base Pairing • DNA Replication Fork is Assymetrical • Replication occurs in 5’ 3’ Direction

  9. Semi-conservative Replication

  10. Semi-conservative Replication

  11. DNA Replication

  12. DNA Replication

  13. Tools of Replication • Enzymes are the tools of replication: • DNA Polymerase - Matches the correct nucleotides then joins adjacent nucleotides to each other • Primase - Provides an RNA primer to start polymerization • Ligase - Joins adjacent DNA strands together (fixes “nicks”)

  14. More Tools of Replication • Helicase - Unwinds the DNA and melts it • Single Strand Binding Proteins - Keep the DNA single stranded after it has been melted by helicase • Gyrase - A topisomerase that Relieves torsional strain in the DNA molecule • Telomerase - Finishes off the ends of DNA strands

  15. In the Beginning… • Problems due to structure • DNA is double-stranded • Template must be single-stranded • Strands must be separated • Separation is difficult due to structure • Melting strands causes tension elsewhere • If unrelieved tension can snap DNA strand

  16. Topoisomerase (Gyrase) • Relieves stress caused by melting DNA • Cleaves DNA and spins around itself to unwind helix • Type I cleaves one strand, type II cleaves both • Reseals DNA strands after relaxation achieved

  17. DNA Replication DNA Helicase • Hydrolyze ATP when bound to ssDNA and opens up helix as it moves along DNA • Moves 1000 bp/sec • 2 helicases: one on leading and one on lagging strand • SSB proteins aid helicase by destabilizing unwound ss conformation

  18. Single Strand Binding Protein • Binds to DNA with no sequence preference • Binds tighter to single strand than double • Keeps separated strands from rejoining

  19. DNA Replication SSB proteins help DNA helicase destabilizing ssDNA

  20. Primase • Creates a primer for DNA polymerase • Template-dependent • An RNA polymerase • Active briefly at beginning of strand synthesis

  21. DNA Replication DNA Primase • uses rNTPs to synthesize short primers on lagging Strand • Primers ~10 nucleotides long and spaced ~100-200 bp • DNA repair system removes RNA primer; replaces it w/DNA • DNA ligase joins Okazaki fragments

  22. DNA Polymerase • Enzyme that synthesizes a DNA strand • Uses existing strand as template • Requires a free “3’ end” to add new nucleotides • Has several catalytic functions • Several forms exist

  23. Eukaryotic DNA Polymerases Enzyme Location Function • Pol  (alpha) Nucleus DNA replication • includes RNA primase activity, starts DNA strand • Pol  (gamma) Nucleus DNA replication • replaces Pol  to extend DNA strand, proofreads • Pol  (epsilon) Nucleus DNA replication • similar to Pol , shown to be required by yeast mutants • Pol  (beta) Nucleus DNA repair • Pol  (zeta) Nucleus DNA repair • Pol  (gamma) Mitochondria DNA replication

  24. Maintenance of DNA Sequences DNA Polymerase as Self Correcting Enzyme • Correct nucleotide has greater affinity for moving polymerase than incorrect nucleotide • Exonucleolytic proofreading of DNA polymerase • DNA molecules w/ mismatched 3’ OH end are not effective templates; polymerase cannot extend when 3’ OH is not base paired • DNA polymerase has separate catalytic site that removes unpaired residues at terminus

  25. Maintenance of DNA Sequences High Fidelity DNA Replication • Error rate= 1 mistake/109 nucleotides • Afforded by complementary base pairing and proof-reading capability of DNA polymerase

  26. DNA Replication DNA Polymerase held to DNA by clamp regulatory protein • Clamp protein releases DNA poly when runs into dsDN • Forms ring around DNA helix • Assembly of clamp around DNA requires ATP hydrolysis • Remains on leading strand for long time; only on lagging strand for short time when it reaches 5’ end of proceeding Okazaki fragments

  27. DNA Replication Okazaki Fragments • RNA that primed synthesis of 5’ end removed • Gap filled by DNA repair enzymes • Ligase links fragments together

  28. DNA Pol. 5’ 3’ 3’ 5’ Okazaki Fragment RNA Primer DNA Pol. 5’ 3’ 3’ 5’ RNA Primer RNA and DNA Fragments 5’ 3’ 3’ 5’ RNA Primer Nick Extension - Okazaki Fragments DNA Polymerase has 5’ to 3’ exonuclease activity. When it sees an RNA/DNA hybrid, it chops out the RNA and some DNA in the 5’ to 3’ direction. DNA Polymerase falls off leaving a nick. Ligase The nick is removed when DNA ligase joins (ligates) the DNA fragments.

  29. DNA ReplicationInitiation and Completion of DNA Replication in Chromosomes • Bacteria • Single Ori • Initiation or replication highly regulated • Once initiated replication forks move at ~400-500 bp/sec • Replicate 4.6 x 106 bp in ~40 minutes

  30. DNA ReplicationInitiation and Completion of DNA Replication in Chromosomes • DNA Replication Begins at Origins of Replication • Positions at which DNA helix first opened • In simple cells ori defined DNA sequence 100-200 bp • Sequence attracts initiator proteins • Typically rich in AT base pairs

  31. DNA ReplicationInitiation and Completion of DNA Replication in Chromosomes • Eukaryotic Chromosomes Have Multiple Origins of Replication • Relication forks travel at ~50 bp/sec • Ea chromosome contains ~150 million base pairs • Replication origins activate in clusters or replication units of 20-80 ori’s • Individual ori’s spaced at intervals of 30,000-300,000 bp

  32. DNA ReplicationInitiation and Completion of DNA Replication in Chromosomes • Mammalian DNA Sequences that Specify Initiation of Replication • 1000’s bp in length • Can function when placed in regions where chromo not too condensed • Human ORC required for replication initiation also bind Cdc6 and Mcm proteins • Binding sites for ORC proteins less specific

  33. The Eukaryotic Problem of Telomere Replication RNA primer near end of the chromosome on lagging strand can’t be replaced with DNA since DNA polymerase must add to a primer sequence.

  34. Different types of Nucleotide Polymerases • DNA polymerase Uses a DNA template to synthesize a DNA strand 2) RNA polymerase Uses a DNA template to synthesize an RNA strand (= transcription) 3) Reverse transcriptase Uses an RNA template to synthesize a DNA strand Found in many viruses Telomerase is a specialized reverse transcriptase

  35. Telomerase • Two components of the human telomerase: • the human RNA subunit(hTR) • 5’-CUAACCCUAAC-3’ • The human telomerase • reverse transcriptase(hTERT)

  36. Telomerase • Function: • Specialized reverse transcriptase • Prevents “shortening ends problem” problem by adding telomeres to the end • Copies only a small segment of RNA that it carries by itself • Requires a 3’ end as a primer • Synthesis proceeds in 5’ – 3’ direction • Synthesizes one repeat then repositions itself • When active provides cell immortality

  37. Telomerase is composed of both RNA and protein

  38. What are telomeres? • Telomeres are… • Repetitive DNA sequences at the ends of all human chromosomes • They contain thousands of repeats of the six-nucleotide sequence, TTAGGG • In humans there are 46 chromosomes and thus 92 telomeres (one at each end)

  39. Telomeres Repeated G rich sequence on one strand in humans: (TTAGGG)n Repeats can be several thousand basepairs long. In humans, telomeric repeats average 5-15 kilobases Telomere specific proteins, eg. TRF1 & TRF2 bind to the repeat sequence and protect the ends Without these proteins, telomeres are acted upon by DNA repair pathways leading to chromosomal fusions

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