Variation in DNA ligase structure during repair and replication processes in monkey kidney cells

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• DNA ligases are crucial enzymes for DNA replication, repair, and recombination.
• Characterizing DNA ligase activity and its associated peptides provides insight into cellular processes.
• SV40 infection and mitomycin C treatment are known to affect cellular DNA metabolism.

Purpose of the Study:

• To characterize the catalytically active DNA ligase peptides in CV1-P monkey kidney cells.
• To investigate the changes in DNA ligase peptide profiles following SV40 infection or mitomycin C treatment.
• To determine if there are common active polypeptides between different forms of DNA ligase.

Main Methods:

• Detection of catalytically active DNA ligase using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) activity gels.
• Purification of DNA ligase using hydroxylapatite column chromatography.
• Analysis of peptide molecular weights (Mr) using SDS-PAGE.

Main Results:

• Two distinct DNA ligase activities, ligase I and ligase II, were identified in control cells.
• Ligase I showed major peptides at 120, 110, 70, and 58 kDa; ligase II showed peptides at 65 and 58 kDa.
• SV40 infection increased 120, 110, 70, and 58 kDa peptides, while mitomycin C increased 70 and 58 kDa peptides and decreased 120 and 110 kDa peptides.
• Autoproteolysis experiments indicated a common 58 kDa peptide in both ligase I and II.

Conclusions:

• The 58 kDa peptide appears to be a common active polypeptide shared between DNA ligase I and II.
• SV40 infection and mitomycin C treatment differentially modulate the expression or stability of DNA ligase peptides.
• These findings suggest a complex regulation of DNA ligase activity in response to cellular stress and viral infection.