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DNA Structure and Replication

DNA Structure and Replication. Pollard & Earnshaw Ch 12-14, 40-42 Structure Chemical composition Filament packing Chromosome organization Replication Origin of replication complex Licensing MCM/polymerase. C. O. Base. OPO 3. C. C. OH. O. C. C. C. OH. C. C. C. OH. OH. OH.

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DNA Structure and Replication

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  1. DNA Structure and Replication • Pollard & Earnshaw Ch 12-14, 40-42 • Structure • Chemical composition • Filament packing • Chromosome organization • Replication • Origin of replication complex • Licensing • MCM/polymerase

  2. C O Base OPO3 C C OH O C C C OH C C C OH OH OH C OH Sugar backbone • Pentose sugar (deoxyribose) • 5’ Phosphate • Nitrogen rich base deoxyribonucleotide Adenosine Ribose

  3. Pyrimidine • Single nitrogen rich ring • Planar bases due to N and O double bonding Thymidine Cytidine

  4. Purine • Double N-rich ring • Also planar • Base pair are asymmetric Guanosine Adenosine

  5. Base Pairing • Hydrogen bonds define pairing T G C A

  6. G-band ideogram karyotype of human chromosomes shows distinct bands Macromolecular conformation • Sequence effects • Accessory proteins • Chromatin • Chromosome • Covalent modification • Specialized domains • Telomere • Centromere

  7. Chromatin: Nucleosome • Histone octamer • Positively charged, attract negative phosphates • Subject to extensive modification • DNA • 166 BP • Twice wrapped

  8. 30 nm fiber • Bead-on-string 10 nm fiber • 166 BP nucleosome • 34 BP linker DNA • Histone H1 • Condenses to 30 nm fiber • 11nm helix • 6 nucleosomes per turn • H4 (deacetylated) Dorigo et al., 2004

  9. Heterochromatin • Transcriptionally inert • Highly condensed • Gene inactivation • Barr body • CG methylation

  10. Further condensation • 100-300 nm fiber • Domain loop model • 50-100 kb • Scaffold/Matrix Association Region • AT rich • Base unpairing region • Nuclear Matrix • DNA topoisomerase: DNA knots • Condensin: supercoiling

  11. Centromere • Chromatid pairing • Kinetochore • CEN sequence • AT and AG Satellites • Epigenetic modification • Unidentified in mammals • CENP proteins

  12. Telomere • Special structure to differentiate from strand break • 600-2500x 5’-CCCTAA-[…]-TTAGGG-3’ • 200 unpaired base overhang • Prevent chromosomal erosion • Telomerase elongates 3’ DNA • Built-in RNA primer • Active in intestines, testis, cancers • Replicative senescence

  13. Telomere structure • Telomere Repeat Factors (TRF) • Ku capping protein (yeast) • Strand repair function • Loop formation • Mammalian • Physical distribution

  14. Generic scheme of template-mediated synthesis • Identify the start site • Assemble the synthetic machinery • Wait for an initiation trigger • Synthesize • Stop/clean-up

  15. Isolation of DNA polymerase • Arthur Kornberg, 1959 Nobel Prize • Work in 1956-1958 with Maurice Bessman, Ernest Simms, I.R. Lehman and Julius Adler • “Vital” processes vs chemical processes • Eduard Buchner, 1907 • Cell-free synthesis of DNA • DNA + cell extract  dinucleotides • NTP + DNA + cell extract  dinuc +NTP

  16. Cell free synthesis of DNA • Highly optimized system • E coli (doubling time 20 minutes) • Massively radioactive NTP • Very short incubation • Assay conversion of acid-soluble NTP to acid-insoluble DNA • NTP + DNA + cell extract 0.0005% DNA

  17. Cellular fractionation • Start with 60L E coli culture 500 g cells • Lyse and extract 40 g protein • Synthesizes 1 nmole DNA/30 min/mg • Mass/density separation • Size separation • Streptomycin precipitation • Precipitates DNA • Extract 2.5% protein • Synthesizes 43 nmole DNA/30 min/mg

  18. Cellular fractionation • DNAse digest • Solubilize DNA-bound protein • 65% protein remains soluble (1.6% of total) • Synthesizes 67 nmole • Alumina gel precipitation • Protein polarity, ala chromatography • Collect 25% of DNAse fraction (0.4% total) • Synthesizes 200 nmole/30 min/mg • Two further fractionations • Around 0.02% starting protein • 2000 nmole/30 min/mg (~20% total activity)

  19. Enzymatic properties • Synthesizes DNA from diverse templates • Bacterial, plant, mammal • DNA is fundamentally identical • Product has same dinucleotide composition • DNA is a template, not a primer • Faster on denatured DNA • ie: single stranded • Further validated Watson & Crick “template” • Requires long strand template • Works much better on phage than genomic

  20. Base OPO3 C O O Base OPO PO3 PO3 C C C O O C C C C C C OH OH DNA Synthesis • DNA polymerase • Deoxyribose 3’ hydroxyl “attacks” nucleoside triphosphate • Forms phosphodiester bond • Leaves HP2O73- • Never backwards • Okazaki fragments

  21. DNA Polymerase Template strand enters polymerase dsDNA exits Pocket for NTP entry http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=69181

  22. DNA Replication • Origin of Replication Complex (ORC) anneals to origin • ORC recruits Mini Chromosomal Maintenance (MCM) • MCM recruits Cdc45p • Cdc45p recruits DNA polymerase a/primase complex • Replication Factor C (RFC) displaces pola • RFC recruits Proliferating Cell Nuclear Antigen (PCNA) • PCNA recruits pold • DNA ligase stitches DNA fragments together

  23. Origin of Replication • Prokaryotes • Single, circular DNA ~4,000,000 bp • Replicator/Autonomously Replicating Sequence • Identification by restriction fragment selection Moore, et al.. Construction of chimeric phages and plasmids containing the origin of replication of bacteriophage lambda. Science (1977) 198:1041-6. • DNA synthesis 1000 bp/s or 65 min replication • Simple Eukaryotes (S cerevisiae) • 16 chromosomes, 107 bp genome • Replicase 1000 bp/min or 10 hour !? replication • ARS • ~400 • 150 bp consensus sequence

  24. Origin of Replication • Multi-cellular Eukaryotes • 2-D electrophoretic fractionation (fig 42-14) • Initiation Zone • Epigenetic mechanisms • Proteins and mechanisms are highly conserved • Kornberg’s templates had to be long in order to include an ORI

  25. Pre-replication complex • Origin Recognition Complex • Assembles at origins during G1 • Orthologous to E Coli DnaA • Cell Division Control 6+Cdt1 • Inhibitory complex • E Coli DnaC • Mini-Chromosomal Maintenance proteins • Recruited by Cdc6+Cdt1 • Licensing agent • E Coli DnaB Sun & al NSMB 2013

  26. Transition to Replication • Cdc45p anneals to ORC & Mcm • Recruit GINS (Sld5, Psf1, Psf2, and Psf3) • Activates Mcm helicase • Recruits DNA polymerases and RPA • RPA ssDNA binding protein Leading strand Laggingstrand Simon & al Nature (2014)

  27. Synthesis • Polymerase a/Primase • Polymerase d & e • Proofreading • DNA polymerase error rate ~1:104-105 • Human genome is 3 109 bases ~10,000 errors per mitosis. • Proliferating Cell Nuclear Antigen (PCNA) error correction 1:109 bases, 3 errors per mitosis • 1013 cells or 43 divisions => 130 base errors per adult genome • Okazaki fragment • Topoisomerase

  28. Summary Cdc6 is believed to remain associated with MCMs Fig 42-11

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