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BIO 404/504 – Molecular Genetics
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  1. BIO 404/504 – Molecular Genetics Dr. Berezney Lecture 1

  2. DNA REPLICATION LECTURES Lecture 1:(1) Fundamentals of DNA Replication: semiconservative replication, bidirectional fork movement, replicons and replicon origins. (2)DNA Replication Mechanisms:unwinding at ori, role of primer, directionality, components and their role in prokaryotes versus eukaryotes, leading versus lagging strand synthesis. [Waga & Stillman, 1994] Lecture 2:Temporal Regulation of DNA Replication in Mammalian Cells: CdK’s and other Factors [Zou and Stillman, 2000] Lecture 3:Assembly, Function & Dynamics of DNA Replication Sites in Cells [Lemon & Grossman, 1998; Sporbert et al., 2002]

  3. “It has not escaped our notice that the specific pairing that we have postulated immediately suggests a possible copying mechanism for the genetic material.” From Watson and Crick, Nature, APRIL 25, 1953

  4. DNA Replication in Both Prokaryotes and Eukaryotes is Bidirectional

  5. DNA Replication of the E. coli Chromosome: A Single Circular Replicon

  6. TIME TO REPLICATE THE GENOME E. Coli Human Bi-directional fork rate 6 Kbp/ min 180 Kbp/ min 4.5 mbp Genome size 6,000 mbp 6,000 mbp / 46 chromosomes (130 mbp each) 130 / 0.006 = 21,666 min = 361 hours = 15 days Replication based on a single origin per chromosome 4.5/0.180 = 25 min 8 hours with ~ 1000 origins (replicons per chromosome) 25-30 min Actual time to replicate the genome

  7. Spatial and Temporal Organization of DNA Replication in Eukaryotic Cells • DNA replication in eukaryotes proceeds bidirectionally at multiple and discontinuous subunits along the chromosomal DNA termed ‘replicons’. • There are approximately 60,000 replicons per mammalian genome with an average size of ~100 kbp. • Groups of adjacent replicons termed ‘replicon clusters’ replicate synchronously during S-phase. • Understanding the spatio-temporal organization of replicon cluster synthesis is key to understanding the overall coordination and choreography of genome replication

  8. Bidirectional Replication: The Step-up/Step-down or Hot/Warm Labeling Technique

  9. Replication Origins in Procaryotic E. coli

  10. The Pol III Holoenzyme is a Dimer

  11. The Pol III Dimeric Holoenzyme at the Replication Fork: Coordination of Leading and Lagging Strand Synthesis

  12. Computer Images of PCNA Trimer (left) and β-subunit Dimer (right) Ring Structures

  13. Leading-Lagging Strand Model for Eukaryotic DNA Replication

  14. Comparing Factors and Events for Lagging Strand Synthesis in E. coli versus Humans

  15. “PRE-WAGA-STILLMAN” • LEADING STRAND: pol α-primase  pol δ [pol switching] • LAGGING STRAND: pol α-primase • “POST-WAGA-STILLMAN” • LEADING STRAND: pol α-primase  pol δ [pol switching] • LAGGING STRAND: pol α-primase  pol δ [pol switching] • WAGA & STILLMAN IN VITRO DNA REPLICATION SYSTEM • pSV011 (SV-40 ori plasmid) • T-antigen • RPA • RFC • pol delta • pol alpha-primase • MF1 (5’-3’ exonuclease) • RNase H • DNA ligase • DNA topoisomerases I and II • dNTP’s with one or more labeled with 32P or chemiluminescence. • COMPLETE REACTION  COMPLETELY REPLICATION DNA PRODUCTS: • Closed circular (FORM I) • Relaxed (nicked) circular (Form II)

  16. Figure 1: Requirement for RFC, PCNA and pol δ for Complete SV40 DNA Replication

  17. Figure 4: Analysis of the products from DNA synthesis reactions by pol δ using the synthetic, lagging-strand template

  18. Figure 5: Analysis of the products from DNA synthesis reactions by pol α/primase using the synthetic, lagging-strand template

  19. LAGGING STRAND LEADING STRAND