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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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Bio 404 504 molecular genetics

BIO 404/504 – Molecular Genetics

Dr. Berezney

Lecture 3: Assembly, Function & Dynamics of Replication Sites in Living Cells



Fig 3: Prokaryotic DNA replication at fixed sites during rapid growth (Dingman, 1974)

Multi-Fork Replication


Visualizing origins of replication in bacillus subtilis

or rapid growth (Dingman, 1974)

Ori

or

Replication complex

DNA

Visualizing origins of replication in Bacillus subtilis

  • Summary of Webb et al., Cell, 1997

    (a) Cassette (multiple tandem repeats) of lac operator inserted near ori.

    (b) Express fusion protein (GFP-lac repressor)

    (c) GFP marks the ori

  • Summary of Lemon & Grossman, Science, 1998

    (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


Growth in Succinate rapid growth (Dingman, 1974)

(E-G)

Figure 1: Localization of replicative DNA polymerase in living cells

(Lemon & Grossman, 1998)

No spots- 25%

Spots-75%

1 spot – 75%

2 spots – 25%

3&4 spots – 0%

Growth in Glucose

(H-I) [Multi-fork Rep]

Growth in Glucose

(A-D)

No spots- 2%

Spots-98%

1 spot – 34%

2 spots – 33%

3 spots – 23%

4 spots – 10%

DnaA - Yes

Τau-GFP

DnaA- No

δ-GFP


Table 1: Distribution of Pol C-GFP foci per cell in various culture conditions (Lemon & Grossman, 1998)


(A) culture conditions (Lemon & Grossman, 1998)

(C)

(D)

(E)

(F)

(H)

(G)

Figures 2 & 3: Model for the localization of the replicative polymerase in B. subtilis(Lemon & Grossman, 1998)

Succinate

Glucose


Fig 4: Fixed Sites for Eukaryotic DNA replication at multiple replicons (DNA loops)(Dingman, 1974)


Hierarchy of Chromatin Organization in the Cell Nucleus: multiple replicons (DNA loops)

Nuclear Matrix Associated Chromatin Loops


Chromatin Organization and Function on the Nuclear Matrix multiple replicons (DNA 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”


Eukaryotic DNA is replicated as multiple replicons (DNA loops)~100

kb units of DNA

termedreplicons

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


(Ma et al., 1998) multiple replicons (DNA loops)

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.


Quantitative Image Analysis 3-D MICROSCOPY & COMPUTER IMAGING

CONTOUR ANALYSIS OF DNA REPLICATION SITES

10

9

11

3

5

1

4

2

Optical section

8

6

7

3-D organization (1000 sites per nucleus)

Number, XY / XYZ Coordinates &

Quantitative co-localization


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


Functional Model of ~1 Mbp Chromatin Domains REPLICATION SITES IN MAMMAMLIAN CELLS

G1

Non-Replicating Chromatin Domain

S

Replication

machinery

Replicating ChromatinDomain

S or G2

Non-ReplicatingChromatin Domains


GFP-PCNA Stable Transfectant REPLICATION SITES IN MAMMAMLIAN CELLSMouse 3T6 Cell Line

Early, Mid and Late S patterns of GFP-PCNAin living cells

Early S Mid S Late S


Sporbert et al 2002 addresses mechanisms of dna replication in living cells using gfp pcna
Sporbert et. al. (2002) Addresses Mechanisms of DNA Replication in Living Cells using GFP-PCNA

  • GLOBAL LEVEL: Does the replication machinery (replication factories) shuttle from one chromatin domain to another or does each replication factory assemble de novo at each chromatin domain?

  • MOLECULAR LEVEL:Is PCNA on the lagging strand cycling on and off the template for each Okazaki fragment in concert with the nucleoplasmic pool of PCNA or is the PCNA fixed at a stable leading/lagging strand replication complex or otherwise confined to the replicating chromatin domain?


Figure 1: GFP-PCNA mimics the endogenous PCNA in binding tightly to replication foci during S phase


Pulse-Chase Experiments: tightly to replication foci during S phaseFigure 5: Spatial- temporal separation of GFP-PCNA from newly replicated DNA

Post-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: tightly to replication foci during S phase 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


Figure 6: Models of PCNA ring dynamics at the replication fork

Stable, dimeric polymerase complex

(No recovery)

Constant assembly of new PCNA rings

(Fast recovery)

Internal recycling by treadmill mechanism

(No recovery)


Conclusions of sporbert et al 2002
Conclusions of Sporbert et. al., 2002 fork

  • Each replication factory (replicational machinery) assembles de novo at each chromatin domain.

  • PCNA at the lagging strand is not cycling on and off with the nucleoplasmic pool of free PCNA. Instead it is either a part of a stable dimeric (leading/lagging strand) replication complex or it is confined to the replicating chromatin domain during cycling.

  • These findings are consistent with DNA replication occurring at replication factories composed of multiple, fixed dimeric and bidirectional replisomes which are assembled onto higher order chromatin domains to initiate DNA replication and are dissassembled when replication of these 1 mbp chromatin domains is completed.


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