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DNA Replication and Repair

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  1. DNA Replication and Repair Monday, August 4

  2. Figure 16.7 A model for DNA replication: the basic concept

  3. Figure 16.7 A model for DNA replication: the basic concept The hydrogen bonds are broken, and the two strands unwind and separate

  4. Figure 16.7 A model for DNA replication: the basic concept

  5. Figure 16.7 A model for DNA replication: the basic concept

  6. Figure 16.8 Three alternative models of DNA replication Dark blue = original parent strand Light blue = newly synthesized strand

  7. Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication Heavy isotopes of nitrogen were used to label the nucleotides which were then incorporated into DNA

  8. Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication Any newly synthesized DNA at this point would incorporate the lighter more common nitrogen isotope

  9. Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication The DNA could then be separated on the basis of density by centrifugation The results from the first replication yielded a band of hybrid (15N-14N) DNA

  10. Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication The results from the second replication yielded a band of hybrid (15N-14N) DNA and one of light (14N) DNA

  11. DNA Replication occurs with remarkable speed and accuracy • Bacteria have a single, circular chromosome • E.coli contains 5 million bps and can replicate and divide is less than an hour • Humans have 46 chromosomes of large molecules of DNA representing about 6 billion bps • Replication of this DNA is achieved in a few hours with very few errors

  12. Figure 16.10 Origins of replication in eukaryotes Replication begins at many specific sites that have a specific sequences recognized by proteins that bind and separate the strands to form replication bubbles Bubbles expand laterally as DNA replication proceeds in both directions (bidirectional) Eventually the bubbles fuse and synthesis of the daughter strands is complete Visualization of 3 replication bubbles along DNA (arrows indicate direction of replication

  13. Figure 16.11 Incorporation of a nucleotide into a DNA strand Rate of elongation: Bacteria = 500 nt/sec Humans = 50 nt/sec DNA polymerase Triphosphate monomers are chemically reactive Exergonic reaction – unstable cluster of negative charges

  14. Figure 16.12 The two strands of DNA are antiparallel DNA strands have polarity: 5’  3’ The phosphate group is attached to the 5’ carbon of the deoxyribose sugar The phosphate group of one nucleotide is attached to the 3’ carbon of the deoxyribose of the adjacent nucleotide 5’-P  3’-OH Antiparallel arrangement: The sugar-phosphate backbones run in opposite directions DNA replication only proceeds in the 5’  3’ direction. DNA polymerases only adds nucleotides to the 3’ end.

  15. Figure 16.13 Synthesis of leading and lagging strands during DNA replication The leading strand is synthesized continuously as the replication fork proceeds. The lagging strand is synthesized in 100-200nts fragments (Okazaki) which are joined by DNA ligase.

  16. Figure 16.14 Priming DNA synthesis with RNA DNA polymerases cannot begin synthesis of polynucleotides, they can only elongate an existing chain bound to the template strand. The start of a new DNA molecule begins with a short (10nt) RNA primer. An enzyme, primase, joins RNA nucleotides to make the primer. Another DNA polymerase can later replace the RNA primer with DNA.

  17. Figure 16.15 The main proteins of DNA replication and their functions Open strands Hold strands apart

  18. Figure 16.16 A summary of DNA replication DNA pol replaces RNA

  19. Figure 16.16 A summary of DNA replication SSB stabilize unwound DNA Helicase unwinds DNA DNA pol replaces RNA

  20. Figure 16.16 A summary of DNA replication Leading strand synthesized 5’3’ SSB stabilize unwound DNA Helicase unwinds DNA DNA pol replaces RNA

  21. Figure 16.16 A summary of DNA replication Leading strand synthesized 5’3’ SSB stabilize unwound DNA Lagging strand synthesized in fragments Helicase unwinds DNA Joins fragments DNA pol replaces RNA

  22. Figure 16.16 A summary of DNA replication Proteins involved in DNA replication form a single, large complex that is stationary during replication. This complex may be anchored to the nuclear matrix (framework of fibers extending through the nucleus). DNA is reeled into the complex and daughter strands are extruded. Leading strand synthesized 5’3’ SSB stabilize unwound DNA Lagging strand synthesized in fragments Helicase unwinds DNA Joins fragments DNA pol replaces RNA

  23. Maintaining the Integrity of DNA • Accuracy in DNA replication is essential to prevent errors or mutations in the newly synthesized DNA • DNA polymerase has proofreading ability • The enzyme checks each nucleotide as it is incorporated into the chain • Any incorrectly paired base is removed by its 3’  5’ exonuclease activity and synthesis is resumed • The error rate of DNA pol is 1 in 10,000 • Cells rely on DNA repair pathways to monitor DNA and correct mismatched bases or repair damaged DNA • 130 DNA repair enzymes have been identified in humans • DNA damage occurs by X-rays, UV, spontaneous chemical changes

  24. DNA Repair • Mismatch Repair • Nucleotide excision repair • Damaged DNA is removed by a nuclease • Gap is filled with correct nucleotides by DNA pol and ligase • Example: thymine dimers • Exposure to UV rays (sun light) can cause the covalent linking of adjacent thymine bases • This creates a kink in the DNA strand which interferes with DNA replication

  25. Figure 16.17 Nucleotide excision repair of DNA damage

  26. Replication Termination • DNA pol can only synthesis 5’  3’ • Incapable of completing the 5’ ends of daughter DNA strands • Repeated rounds of replication result in shortening DNA molecules • Solution: telomeres • chromosomal DNA has special nucleotide sequences at their ends consisting of multiple repetitions (100-1000) of one short sequence: TTAGGG • Telomeres are restored by telomerase, which catalyzes the lengthening of these sequences

  27. Figure 16.18 The end-replication problem

  28. Replication Termination • DNA pol can only synthesis 5’  3’ • Incapable of completing the 5’ ends of daughter DNA strands • Repeated rounds of replication result in shortening DNA molecules • Solution: telomeres • chromosomal DNA has special nucleotide sequences at their ends consisting of multiple repetitions (100-1000) of one short sequence: TTAGGG • Telomeres are restored by telomerase, which catalyzes the lengthening of these sequences

  29. Figure 16.19a Telomeres and telomerase: Telomeres of mouse chromosomes

  30. Figure 16.19b Telomeres and telomerase Telomerase is a complex of protein and RNA: RNA contains a sequence that serves as the template for new telomere segments Telomerase is not present in somatic cells – telomeres are shorter in older individuals Telomerase is present in germ-line cells and cancerous cells