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In vitro rabbit mammary gland chromatin replication studied by micrococcal nuclease digestion
Abstract:
Isolated nuclei from pregnant rabbit mammary glands were labelled with [3H]dTTP under conditions for DNA synthesis and subsequently digested with micrococcal nuclease. Replicating chromatin was found to exhibit increased susceptibility towards the nuclease. Analysis of chromatin digestion products by sucrose density gradient centrifugation demonstrated the association of in vitro replicated DNA with nucleosomes. Furthermore, the distribution of DNA polymerizing activity was studied in isolated nuclease-digested mammary gland chromatin. About 90% of all recovered nuclear DNA polymerizing activity cosedimented with nucleosomal particles, mainly with mononucleosomes. The distribution of DNA polymerases alpha and beta in chromatin isolated from the mammary glands of pregnant and lactating rabbits was compared. In these physiological states the mononucleosome-associated DNA polymerase alpha activity varied in accordance with the rate of DNA synthesis.
Insights
Replicating chromatin in rabbit mammary glands is more susceptible to micrococcal nuclease. DNA synthesis activity, particularly DNA polymerase alpha, is associated with nucleosomes during DNA replication.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Chromatin structure plays a crucial role in regulating DNA replication.
- Understanding the association of DNA polymerases with chromatin is essential for elucidating replication mechanisms.
Purpose of the Study:
- To investigate the susceptibility of replicating chromatin to nuclease digestion.
- To determine the association of newly synthesized DNA and DNA polymerizing activity with nucleosomes.
- To compare the distribution of DNA polymerases alpha and beta in pregnant and lactating rabbit mammary gland chromatin.
Main Methods:
- Isolation of nuclei from pregnant rabbit mammary glands.
- Labeling of DNA with [3H]dTTP under DNA synthesis conditions.
- Digestion of chromatin with micrococcal nuclease.
- Analysis of digestion products using sucrose density gradient centrifugation.
- Assay of DNA polymerizing activity and DNA polymerases alpha and beta.
Main Results:
- Replicating chromatin showed increased susceptibility to micrococcal nuclease digestion.
- In vitro replicated DNA was found associated with nucleosomes.
- Approximately 90% of nuclear DNA polymerizing activity cosedimented with nucleosomal particles, primarily mononucleosomes.
- Mononucleosome-associated DNA polymerase alpha activity correlated with the rate of DNA synthesis in pregnant and lactating rabbits.
Conclusions:
- Replicating chromatin exhibits altered structural properties making it more accessible to nucleases.
- DNA replication machinery, including DNA polymerases, is closely associated with nucleosomes.
- DNA polymerase alpha activity is dynamically regulated and linked to DNA synthesis rates in the mammary gland during pregnancy and lactation.