Characterisation of poly(ADP-Rib) polymerase activity in nuclei from the slime mould Dictyostelium discoideum

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Poly(ADP-Rib) synthesis is a crucial post-translational modification involved in DNA repair and gene regulation.
• Understanding the enzymes responsible for this process is vital for comprehending cellular responses to stress.

Purpose of the Study:

• To characterize the poly(ADP-Rib) polymerase activity in isolated nuclei of Dictyostelium discoideum.
• To determine the optimal in vitro conditions for enzyme activity and its kinetic properties.

Main Methods:

• Isolation of nuclei from Dictyostelium discoideum.
• In vitro incubation of nuclei with 3H-NAD to measure poly(ADP-Rib) synthesis.
• Determination of enzyme kinetics, including Km and inhibition by nicotinamide.

Main Results:

• Isolated D. discoideum nuclei synthesize poly(ADP-Rib) when incubated with 3H-NAD.
• Optimal incubation conditions for the poly(ADP-Rib) polymerase were established.
• The enzyme exhibited a Michaelis constant (Km) of 18 microM for NAD and was inhibited by nicotinamide.
• The majority of synthesized poly(ADP-Rib) was found to be attached to nuclear proteins.

Conclusions:

• Dictyostelium discoideum possesses a functional poly(ADP-Rib) polymerase within its nuclei.
• The enzyme's kinetic properties and substrate specificity were elucidated.
• These findings contribute to the understanding of poly(ADP-Rib) metabolism in this model organism.