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Nucleoids, a subnuclear system capable of chain elongation
J M Collins1, S H Wood, A K Chu
1Department of Biochemistry and Molecular Biophysics, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond.
Biochimica Et Biophysica Acta
|December 22, 1989
Summary
Nucleoids and nuclear matrices differ in DNA chain length and synthesis capabilities. Nuclear matrices show reduced DNA binding but enhanced in vitro synthesis compared to nucleoids.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Nucleoids and nuclear matrices are residual nuclear structures obtained after salt extraction.
- These structures differ based on whether nuclei are digested with DNase prior to extraction.
- Understanding their distinct properties is crucial for studying DNA replication and nuclear organization.
Purpose of the Study:
- To compare the biochemical and functional properties of nucleoids and nuclear matrices.
- To investigate differences in DNA chain length, enzyme binding, and in vitro DNA synthesis.
- To elucidate the role of nuclear extracts in restoring DNA synthesis capabilities.
Main Methods:
- Preparation of nucleoids from non-DNase-digested nuclei via salt extraction.
- Preparation of nuclear matrices from DNase-digested nuclei via salt extraction.
- Analysis of DNA chain length (Svedberg units), bound DNA polymerase alpha and primase, and in vitro DNA synthesis and priming activity.
- Assessment of DNA synthesis elongation after extraction with increasing salt concentrations.
- Restoration of DNA synthesis by adding nuclear extracts.
Main Results:
- Nucleoids contained larger DNA chains (110 S) than nuclear matrices (30 S).
- Nuclear matrices showed less bound DNA polymerase alpha and primase but greater in vitro DNA synthesis and priming.
- Both structures synthesized 4.5 S Okazaki fragments, but DNase-digested nucleoids lost elongation ability above 0.1-0.2 M NaCl, unlike undigested nuclei (up to 0.9 M NaCl).
- Chain elongation to 28 S DNA chains was restorable in nucleoids, but not nuclear matrices, with nuclear extracts.
Conclusions:
- Nucleoids and nuclear matrices exhibit distinct characteristics regarding DNA integrity and replication machinery.
- DNase digestion significantly impacts the salt extractability and DNA synthesis potential of nuclear structures.
- Nuclear extracts contain factors essential for restoring DNA chain elongation, highlighting the dynamic nature of nuclear components.