A cellular single-stranded DNA-dependent ATPase associated with simian virus 40 chromatin

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• SV40 chromatin contains associated proteins, including potential DNA-modifying enzymes.
• ATPase activities are crucial for DNA replication and metabolism.

Purpose of the Study:

• To isolate and characterize a single-stranded DNA-dependent ATPase from monkey kidney cells.
• To differentiate this ATPase from the viral T-antigen ATPase.
• To investigate the enzyme's role in SV40 chromatin.

Main Methods:

• Isolation and biochemical characterization of ATPase from CV-1 cell nuclei.
• Enzyme assays measuring ATP hydrolysis dependent on DNA structure and divalent cations.
• Analysis of substrate specificity using various forms of SV40 DNA.
• Glycerol gradient sedimentation to assess DNA-binding affinity.

Main Results:

• A single-stranded DNA-dependent ATPase was isolated from uninfected monkey kidney cells.
• The enzyme's properties (Km for ATP, phosphocellulose elution, DNA stimulation) distinguish it from SV40 T-antigen.
• ATP hydrolysis requires single-stranded DNA and a divalent cation, with specific kinetic parameters identified.
• The ATPase exhibits high affinity for single-stranded DNA and preferentially binds superhelical Form I SV40 DNA.

Conclusions:

• The identified ATPase is distinct from SV40 T-antigen.
• The enzyme's preference for single-stranded DNA suggests a role in DNA metabolism.
• Hypothesized to bind to single-stranded regions of replication forks within SV40 chromatin.