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Updated: Aug 15, 2026
Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement
Published on: January 19, 2017
Differential replication capacities of G1 and S-phase extracts from sea urchin eggs
1Department of Anatomy and Cellular Biology, Harvard Medical School, Boston, MA 02115.
Abstract:
Sea urchin eggs are arrested in G1 of the first mitotic cell cycle. Fertilization triggers release from G1 arrest and the onset of DNA synthesis about 20 minutes later, even when protein synthesis is blocked. Here we describe extracts from eggs and S-phase embryos that reproduce this stage-specific pattern of DNA synthesis. Fertilized egg extracts formed nuclear membranes around decondensed Xenopus sperm chromatin whereas unfertilized egg extracts did not. Aphidicolin-sensitive deoxynucleotide incorporation was high in extracts of fertilized S-phase eggs and low in those of unfertilized eggs. In contrast, single-stranded DNA templates directed high rates of incorporation in both unfertilized and fertilized egg extracts, suggesting that the stage-specific activities in nuclear DNA synthesis is restricted to initiation on double-stranded DNA. Mixing experiments showed that unfertilized eggs do not contain a dominant inhibitor of replication, nor does fertilization induce the appearance of a soluble, dominant activator.
Insights
Fertilization triggers DNA synthesis in sea urchin eggs by enabling nuclear membrane formation and activating replication on double-stranded DNA. This process is not inhibited by blocking protein synthesis.
Area of Science:
- Cell Biology
- Developmental Biology
- Molecular Biology
Background:
- Sea urchin eggs are arrested in the G1 phase of the cell cycle.
- Fertilization initiates DNA synthesis approximately 20 minutes post-fertilization, independent of protein synthesis.
Purpose of the Study:
- To investigate the molecular mechanisms underlying the initiation of DNA synthesis upon fertilization in sea urchin eggs.
- To characterize the stage-specific DNA synthesis activities in egg and embryonic extracts.
Main Methods:
- Preparation of egg and S-phase embryonic extracts from sea urchins.
- Assay of deoxynucleotide incorporation into DNA using decondensed Xenopus sperm chromatin as a template.
- Use of aphidicolin to assess DNA polymerase activity.
- Employing single-stranded and double-stranded DNA templates.
- Conducting mixing experiments with egg extracts.
Main Results:
- Fertilized egg extracts formed nuclear membranes around sperm chromatin, unlike unfertilized egg extracts.
- Aphidicolin-sensitive deoxynucleotide incorporation was significantly higher in fertilized S-phase egg extracts compared to unfertilized extracts.
- High rates of deoxynucleotide incorporation occurred on single-stranded DNA templates in both extract types.
- Mixing experiments ruled out dominant inhibitors or soluble activators in unfertilized eggs or upon fertilization.
Conclusions:
- The stage-specific activity regulating nuclear DNA synthesis is primarily associated with the initiation of replication on double-stranded DNA.
- Fertilization induces changes that permit DNA replication initiation, likely involving nuclear envelope formation.
- The regulation of DNA replication initiation is intrinsic to the egg and is modulated by fertilization events.
