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DNA polymerase-primase complex in wild-type and ts A1S9 mouse L-cells, temperature-sensitive for DNA replication
Journal of Cellular Physiology
|September 1, 1990
Summary
Temperature-sensitive A1S9 cells show DNA replication arrest due to DNA polymerase-alpha inactivation, not reduced synthesis, impacting cell division.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Temperature-sensitive (ts) mutant cells (ts A1S9) derived from wild-type (WT-4) mouse L-cells exhibit defects in DNA synthesis and cell division at nonpermissive temperatures.
- Understanding the molecular basis of DNA replication arrest in these cells is crucial for elucidating DNA replication mechanisms.
Purpose of the Study:
- To determine the cause of DNA replication arrest in ts A1S9 cells at the nonpermissive temperature.
- To investigate the temperature-induced modifications affecting the activity and synthesis of DNA polymerase-alpha and DNA primase.
Main Methods:
- Comparison of DNA polymerase-alpha and DNA primase activities in ts A1S9 and WT-4 cells at different temperatures over time.
- Immunoprecipitation of [35S]-labeled cell extracts using a monoclonal antibody against DNA polymerase-alpha to analyze protein synthesis and complex structure.
Main Results:
- At the nonpermissive temperature (38.5°C), ts A1S9 cells showed a 90% inhibition of DNA polymerase-alpha activity and undetectable primase activity after 47 hours.
- While synthesis of the 186 kDa subunit of DNA polymerase-alpha was not decreased, its activity was temperature-inactivated.
- A decline in 60 and 48 kDa polypeptides associated with primase activity was observed, alongside an increase in 220 and 186 kDa polypeptides linked to polymerase-alpha activity.
Conclusions:
- The cell cycle arrest and DNA synthesis inhibition in ts A1S9 cells at the nonpermissive temperature are attributed to the temperature-induced inactivation of DNA polymerase-alpha and alterations in DNA primase components.
- Findings support models of coordinated DNA synthesis at the replication fork by polymerase-alpha and polymerase-delta complexes.
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