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The microarchitecture of DNA replication domains
Anatoly A Philimonenko1, Zdenek Hodný, Dean A Jackson
1Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20, Prague 4-Krc, Czech Republic.
Histochemistry and Cell Biology
|October 26, 2005
Summary
Two types of DNA replication factories, replication bodies (RB) and replication foci (RF), were identified in HeLa cells. While both sites contain replication and cell cycle proteins, RB exhibit distinct microarchitecture and protein organization, suggesting functional differences.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- DNA synthesis occurs in discrete nuclear foci within HeLa cells.
- These sites, termed replication factories, can be visualized using biotin-dUTP labeling and electron microscopy.
- Two distinct types, replication bodies (RB) and replication foci (RF), are distinguishable at the ultrastructural level.
Purpose of the Study:
- To investigate the protein composition of RB and RF.
- To determine if differences in protein content correlate with structural and functional distinctions between these replication sites.
Main Methods:
- HeLa cells were permeabilized and allowed to elongate nascent DNA with biotin-dUTP.
- Biotinylated nascent DNA was immunolabeled with gold particles for electron microscopy.
- Double immunogold labeling was employed to analyze the protein composition of RB and RF.
Main Results:
- Both RB and RF contain DNA replication proteins (DNA polymerase alpha, PCNA) and cell cycle regulators (cyclin A, cdk2).
- Both sites also include RNA processing components (Sm, SS-B/La, p80-coilin, hnRNPs A1, C1/C2).
- RB and RF differ in the presence of specific proteins (Cdk1, cyclin B1, PML, lamin B1) and exhibit distinct structural organization, with RB showing more developed microarchitecture and less DNA content.
Conclusions:
- Replication factories (RB and RF) share common protein components but also possess unique protein sets.
- Replication bodies (RB) possess a more complex microarchitecture, suggesting specialized functional roles.
- The observed differences imply that RB and RF may replicate distinct regions of the genome.