BIO 404/504 – Molecular Genetics. Dr. Berezney Lecture 3: Assembly, Function & Dynamics of Replication Sites in Living Cells. Fig 2: DNA replication at fixed sites in prokaryotes during slow growth (Dingman, 1974).
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Lecture 3: Assembly, Function & Dynamics of Replication Sites in Living Cells
Fig 2: DNA replication at fixed sites in prokaryotes during slow growth (Dingman, 1974)
Fig 3: Prokaryotic DNA replication at fixed sites during rapid growth (Dingman, 1974)
DNAVisualizing origins of replication in Bacillus subtilis
(a) Cassette (multiple tandem repeats) of lac operator inserted near ori.
(b) Express fusion protein (GFP-lac repressor)
(c) GFP marks the ori
(a) Visualize replication sites (RS) in B.subtilis using GFP-pol c (III)
construct i.e, tracking RS via pol C in living cells.
(b) At slow growth :
Mobile replication complex – usually two sites at random positions
Fixed replication complex – usually one site at set position
Figure 1: Localization of replicative DNA polymerase in living cells
(Lemon & Grossman, 1998)
No spots- 25%
1 spot – 75%
2 spots – 25%
3&4 spots – 0%
Growth in Glucose
(H-I) [Multi-fork Rep]
Growth in Glucose
No spots- 2%
1 spot – 34%
2 spots – 33%
3 spots – 23%
4 spots – 10%
DnaA - Yes
Table 1: Distribution of Pol C-GFP foci per cell in various culture conditions (Lemon & Grossman, 1998)
Figures 2 & 3: Model for the localization of the replicative polymerase in B. subtilis(Lemon & Grossman, 1998)
Fig 4: Fixed Sites for Eukaryotic DNA replication at multiple replicons (DNA loops)(Dingman, 1974)
Nuclear Matrix Associated Chromatin Loops
Chromatin loops (50-250 Kbp) are attached to nuclear matrix
These chromatin loops are believed to be the fundamental functional units for replication of DNA (replicons) and for the transcription of genes
The machinery for DNA replication and RNA transcription are assembled at the base of the chromatin loops which are attached to the nuclear matrix.
Discrete Sites of DNA replication or transcription have been visualized in the cell nucleus using fluorescence microscopic imaging approaches and are commonly referred to as “DNA replication or transcription factories”
kb units of DNA
Factory Model of DNA Replication
This model proposes that each replisome drives a bidirectional replication forkfixed to the nuclear matrix. Multiple replisomes then cluster together into discrete DNA replicationsites (RS)or “replicationfactories”(RF).
bidirectional replication fork
The experiments of Ma et al. were designed to directly test the Replication Factory Model by determining the number of Replication Sites (RS) and the average lifetime of each RS. This enables calculation of the approximate average amount of DNA and the minimal number of replicons contained in each RS based on the average bidirectional fork rate.
ANALYZING DNA REPLICATION SITES (RS) IN THE CELL NUCLEUS BY 3-D MICROSCOPY & COMPUTER IMAGING
Single Halogenated Nucleoside Labeling Experimentto Determine the Total Number of RS(Ma et al., 1998)
1.Mammalian cells are grown on cover slips and synchronized in early S-phase.
2. Pulse with halogenated nucleoside e.g., 5 min, bromodeoxyuridine (BrdU).
3. Fix cells and label with anti BrdU, and a 20 Ab with FITC (green).
4. Collect optical sections by confocal microscopy.
5. Do computer imaging contour analysis of the individual RS and 3-D reconstruction of the optical sections.
6. Determine the average number of RS in early S phase at any moment of time and the x,y,z coordinates and volumes of all the individual sites.
CONTOUR ANALYSIS OF DNA REPLICATION SITES
3-D organization (1000 sites per nucleus)
Number, XY / XYZ Coordinates &
MAJOR CONCLUSIONS OF MICROSCOPY/IMAGE ANALYSIS OF DNA REPLICATION SITES IN MAMMAMLIAN CELLS
(Ma et al., J.Cell. Biol. 143 (1998) 1415-1425)
There is an average of approximately 1,000 replication sites (RS) active at any moment in early S phase.
Average life-time of an early S RS is about 45 min and contains ~ 1 mbp of DNA organized into at least 6 replicons (chromatin loops).
The RS persist throughout the cell cycle and in future cell generations as ~1 mbp higher order chromatin domains
Non-Replicating Chromatin Domain
S or G2
Early, Mid and Late S patterns of GFP-PCNAin living cells
Early S Mid S Late S
Figure 1: GFP-PCNA mimics the endogenous PCNA in binding tightly to replication foci during S phase
Pulse-Chase Experiments: Figure 5: Spatial- temporal separation of GFP-PCNA from newly replicated DNA
A- 3 min BrdU pulse
C- 10 min BrdU pulse
E- 20 min BrdU pulse
Nascent DNA (3 min pulse)
A- 0 min chase
B- 10 min chase
D- 20 min chase
F- 45 min chase
Photobleaching Experiments: Fluorescence Recovery After Photobleaching (FRAP): Measure the return of fluorescence to a bleached spot
Figure 2: Photobleaching of GFP-PCNA at replication foci does not alter replicational activity or impair de novo assembly of GFP-PCNA
Fig 3: PCNA and RPA 34, two factors involved in DNA replication, show different recovery behavior at replication sites
Figure 4 : PCNA is not directly recycled to newly activated adjacent replication foci
Figure 4: Models for assembly of replication factors at adjacent replication foci or factories (RFs)
Models I and III: Pre-existing replication factories move to new chromatin domains (or chromatin “moves” to RFs)
Models II and IV: Disassembly of replication factories at the end of replication and de novo reassembly at new chromatin domains
Stable, dimeric polymerase complex
Constant assembly of new PCNA rings
Internal recycling by treadmill mechanism