1. BIO 404/504 – Molecular Genetics Dr. Berezney Lecture 3: Assembly, Function & Dynamics of Replication Sites in Living Cells

3. Fig 2: DNA replication at fixed sites in prokaryotes during slow growth (Dingman, 1974)

4. Fig 3: Prokaryotic DNA replication at fixed sites during rapid growth (Dingman, 1974) Multi-Fork Replication

5. 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 or Replication complex DNA or Ori

6. Figure 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-GFP δ-GFP No 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 - Yes DnaA- No

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

8. Figures 2 & 3: Model for the localization of the replicative polymerase in B. subtilis (Lemon & Grossman, 1998) (E) (A) (D) (F) (H) (C) (G) SuccinateGlucose

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

10. Hierarchy of Chromatin Organization in the Cell Nucleus: Nuclear Matrix Associated Chromatin Loops

11. Chromatin Organization and Function on the Nuclear Matrix 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”

12. 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 fork Eukaryotic DNA is replicated as ~100 kb units of DNA termed replicons

13. (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.

14. 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 2 0 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.

15. 1 2 3 4 5 911 10 8 7 6 Optical section Number, XY / XYZ Coordinates & Quantitative co-localization 3-D organization (1000 sites per nucleus) Quantitative Image Analysis CONTOUR ANALYSIS OF DNA REPLICATION SITES

16. 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

17. Functional Model of ~1 Mbp Chromatin Domains G1 Non-Replicating Chromatin Domain S Replicating Chromatin Domain S or G2 Non-Replicating Chromatin Domains Replication machinery

18. Early S Mid S Late S GFP-PCNA Stable Transfectant Mouse 3T6 Cell Line Early, Mid and Late S patterns of GFP-PCNA in living cells

20. 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?

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

22. Nascent DNA (3 min pulse) A- 0 min chase B- 10 min chase D- 20 min chase F- 45 min chase Pulse-Chase Experiments: Figure 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

23. Photobleaching Experiments: Fluorescence Recovery After Photobleaching (FRAP): Measure the return of fluorescence to a bleached spot

24. Figure 2: Photobleaching of GFP-PCNA at replication foci does not alter replicational activity or impair de novo assembly of GFP-PCNA

25. Fig 3: PCNA and RPA 34, two factors involved in DNA replication, show different recovery behavior at replication sites

26. Figure 4 : PCNA is not directly recycled to newly activated adjacent replication foci

27. 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

28. 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)

29. Conclusions of Sporbert et. al., 2002 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.