UB BIO 404 - Lecture 3-Assembly, Function & Dynamics of Replication Sites in Living Cells

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BIO 404/504 – Molecular GeneticsSlide 2Slide 3Slide 4Visualizing origins of replication in Bacillus subtilisSlide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Sporbert et. al. (2002) Addresses Mechanisms of DNA Replication in Living Cells using GFP-PCNASlide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Conclusions of Sporbert et. al., 2002BIO 404/504 – Molecular Genetics Dr. BerezneyLecture 3: Assembly, Function & Dynamics of Replication Sites in Living CellsFig 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) Multi-Fork ReplicationVisualizing 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 positionsFixed replication complex – usually one site at set position orReplication complexDNAorOriFigure 1: Localization of replicative DNA polymerase in living cells (Lemon & Grossman, 1998) Growth in Glucose(A-D) Growth in Succinate(E-G)Growth in Glucose(H-I) [Multi-fork Rep]Τau-GFPδ-GFPNo spots- 25%Spots-75%1 spot – 75%2 spots – 25%3&4 spots – 0%No spots- 2%Spots-98%1 spot – 34%2 spots – 33%3 spots – 23%4 spots – 10%DnaA - YesDnaA- NoTable 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) (E)(A)(D) (F) (H)(C) (G)Succinate GlucoseFig 4: Fixed Sites for Eukaryotic DNA replication at multiple replicons (DNA loops) (Dingman, 1974)Hierarchy of Chromatin Organization in the Cell Nucleus: Nuclear Matrix Associated Chromatin LoopsChromatin Organization and Function on the Nuclear Matrix Chromatin loops (50-250 Kbp) are attached to nuclear matrixThese chromatin loops are believed to be the fundamental functional units for replication of DNA (replicons) and for the transcription of genesThe 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”Factory Model of DNA Replication This model proposes that each replisome drives a bidirectional replication fork fixed to the nuclear matrix. Multiple replisomes then cluster together into discrete DNA replication sites (RS) or “replication factories” (RF).bidirectional replication forkEukaryotic DNA is replicated as ~100 kb units of DNA termed replicons(Ma et al., 1998)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 Experiment to 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.??123459 1110876Optical sectionNumber, XY / XYZ Coordinates &Quantitative co-localization3-D organization (1000 sites per nucleus) Quantitative Image AnalysisCONTOUR ANALYSIS OF DNA REPLICATION SITESMAJOR 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 domainsFunctional Model of ~1 Mbp Chromatin DomainsG1Non-Replicating Chromatin DomainSReplicating Chromatin DomainS or G2Non-Replicating Chromatin DomainsReplicationmachineryEarly S Mid S Late SGFP-PCNA Stable Transfectant Mouse 3T6 Cell LineEarly, Mid and Late S patterns of GFP-PCNA in living cellsSporbert 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 phaseNascent DNA (3 min pulse)A- 0 min chaseB- 10 min chaseD- 20 min chaseF- 45 min chasePulse-Chase Experiments: Figure 5: Spatial- temporal separation of GFP-PCNA from newly replicated DNAPost-Replicated DNAA- 3 min BrdU pulseC- 10 min BrdU pulseE- 20 min BrdU pulsePhotobleaching Experiments: Fluorescence Recovery After Photobleaching (FRAP): Measure the return of fluorescence to a bleached spotFigure 2: Photobleaching of GFP-PCNA at replication foci does not alter replicational activity or impair de novo assembly of GFP-PCNAFig 3: PCNA and RPA 34, two factors involved in


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UB BIO 404 - Lecture 3-Assembly, Function & Dynamics of Replication Sites in Living Cells

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