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BIO 404/504 – Molecular Genetics. Dr. Berezney Lecture 1. DNA REPLICATION LECTURES. Lecture 1: (1) Fundamentals of DNA Replication : semiconservative replication, bidirectional fork movement, replicons and replicon origins .

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dna replication lectures
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]

slide4

“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

slide10

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

slide11

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
slide18

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

slide23

“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)
slide24

Figure 1: Requirement for RFC, PCNA and

pol δ for Complete SV40 DNA Replication

slide27

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

slide28

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

slide30

LAGGING STRAND

LEADING STRAND

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