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DNA Replication

DNA Replication. AHMP 5406. Objectives:. Outline the mechanisms of eukaryotic DNA replication Describe the cellular mechanisms that help avoid error generation during DNA synthesis Describe the possible pathways of DNA repair Relate chromatin density and the cell cycle to DNA replication.

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DNA Replication

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  1. DNA Replication AHMP 5406

  2. Objectives: • Outline the mechanisms of eukaryotic DNA replication • Describe the cellular mechanisms that help avoid error generation during DNA synthesis • Describe the possible pathways of DNA repair • Relate chromatin density and the cell cycle to DNA replication

  3. DNA Replication • The process of copying DS DNA by templated polymerization • In Eukaryotes occurs only during S phase • Overall replication scheme similar to prokaryotes

  4. DNA Replication • Base pairing is responsible for DNA replication and repair • Multiple initiation points • Linear chromosome (Proks. circular) • Many polymerases and accessory factors required

  5. Chromosome Size and Topology

  6. DNA replication is semi-conservative • During one round of replication • One strand used as template

  7. Repl. begins at specific chromosomal sites • Replication origins • Regardless of organism are: • unique DNA segments with multiple short repeats • recognized by multimeric origin-binding proteins • usually contain an A-T rich stretch

  8. Most DNA replication is bidirectional

  9. Eukaryotic Chromosome Replication • DNA replication are very similar in proks and euks • Differences: • Euks have many chromosomes • one in prokaryotes • The problem with nucleosomes • euk DNA is “packaged” • wrapped around histones • In eukaryotes DNA and histones must be doubled with each cell division

  10. Eukaryotic Replication • DNA synthesis • In eukaryotes • small portion of the cell cycle (S) • continuously in prokaryotes • Eukaryotes have more DNA to replicate • How is this accomplished? • Multiple origins of replication • prokaryotes one origin – OriC • Two different polymerases

  11. Problems that must be overcome for DNA polymerase to copy DNA • DNA polymerases can’t melt duplex DNA • Must be separated for copying • DNA polymerases can only elongate a preexisting DNA or RNA strand (the primer) • Strands in the DNA duplex are opposite in chemical polarity • All DNA polymerases catalyze nucleotide addition at 3-hydroxyl end • Strands can grow only in the 5 to 3 direction

  12. Structure of DNA Rep. Fork • Both daughter strands polymerized in 5’-3’ direction • Lagging strand DNA synth. in short segments • Okazaki fragments

  13. Proteins at the fork form a replication machine • Mammalian replication fork

  14. Specialized enzymes • Helicases separate two parental DNA strands • Polymerases synthesize primers and DNA • Accessory proteins promote tight binding of enzymes to DNA • Increase polymerase speed and efficiency (sliding clamp) • Editing exonucleases work with polymerases • Topoisomerases convert supercoiled DNA to the relaxed form

  15. DNA Helicase • Hexameric ring • Separate DNA strands • Use ATP hydrolysis for Energy

  16. Primase • Activated by helicase • Synthesizes short RNA primer • Uses DNA as template

  17. Sliding clamp • Keeps DNA polymerases attached to DNA strand • Assisted by clamp loader through ATP hydrolysis • Will disassociate if DNA pol reaches DS DNA

  18. Single stranded binding proteins • Bind tightly and cooperatively to SS DNA • Do not cover bases • Remain available for templating • Aid in stabilizing unwound DNA • Prevent hairpin structures

  19. Mammalian DNA polymerases • Synthesize new DNA strand • Requires primer • DNA Pol a • Associated with primase • DNA Pol d • Elongates

  20. Mammalian DNA Polymerases • a : Repair and Replication and primase function • b: Repair function • g : Mitochondrial DNA polymerase • d : Replication with PCNA (processivity factor) • e : Replication

  21. Topoisomerase • Some proteins change topology of DNA • Helicase can unwind the DNA duplex • induce formation of supercoils • Topoisomerases catalyze addition or removal of supercoils

  22. Topoisomerase • Type I topoisomerase relax DNA by nicking and closing one strand of duplex DNA • Covalently attach to DNA phosphate • Allow rotation

  23. Topoisomerase • Type II topoisomerase change DNA topology by breaking and rejoining double stranded DNA

  24. Action of E coli Topoisomerase I

  25. Type II topoisomerases (gyrases) change DNA topology by breaking and rejoining double-stranded DNA

  26. Replicated circular DNA molecules are separated by type II topoisomerases Linear daughter chromatids also are separated by type II topoisomerases

  27. The eukaryotic replication machinery is generally similar to that of E. coli

  28. More on Telomeres

  29. Telomeres • Further evidence of a relationship b/w telomere length and aging in humans • Disorder called progerias (premature aging) • Hutchinson-Gilford Syndrome (severe) – death in the teen years • Werner Syndrome (less severe) – death usually in the 40s

  30. Telomere Replication • Regions of DNA at each end of a linear chromosome • Required for replication and stability of that chromosome. • Human somatic cells (grown in culture) divide only a limited number of times (20-70 generations)

  31. Telomere Replication • Correlation between telomere length and the number of cell divisions preceding senescence and death • Cells with longer telomeres survive longer (more divisions) than cells with short telomeres

  32. Problem with Telomeres • DNA polymerase require free 3’OH end • cannot replace the RNA primer • at the terminus of the lagging strand. • If not remedied, the DNA would become shorter and shorter • Telomerase resolves the terminal primer problem

  33. Telomerase • Telomerase = enzyme made up of both protein and RNA • RNA component is base sequence complementary to telomere repeat unit • Catalyzes synthesis of new DNA using RNA as template

  34. 5 3 5 3 End-Replication Problem 5 3 + 5 3 Process Okazaki Fragments 5 3 + 5 3

  35. Telomere Structure 5 3 G-rich C-rich • Telomeres composed of short (6-10 bp) repeats • G-rich in one strand, C-rich in other

  36. Telomerase • Germ-line cells possess telomerase activity • Most human somatic cells lack telomerase activity • Cultured immortal cell lines have been shown to have telomerase activity • Possible cancer therapy may be to control telomerase activity in cancer cells

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