[Conformation limited nucleoside-5'-phosphates as termination substrates for DNA-polymerases]

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Nucleoside analogues are crucial for antiviral and anticancer therapies.
• Understanding DNA polymerase mechanisms requires specific inhibitors and terminators.
• Limited conformational analogues offer unique substrate properties.

Purpose of the Study:

• To investigate the DNA synthesis termination capabilities of novel nucleoside 5 -triphosphate analogues.
• To evaluate these analogues across a range of prokaryotic and eukaryotic DNA polymerases.
• To identify analogues with specific termination profiles.

Main Methods:

• Enzymatic assays using purified DNA polymerases (calf thymus DNA polymerase alpha, terminal deoxynucleotidyl transferase, rat liver DNA polymerase beta, E. coli DNA polymerase I Klenow's fragment, AMV reverse transcriptase).
• Synthesis and characterization of nucleoside 5 -triphosphate analogues with restricted carbohydrate moieties.
• Analysis of DNA synthesis termination and nucleotide incorporation.

Main Results:

• Lyxoanhydronucleoside 5 -triphosphates terminated DNA synthesis catalyzed by reverse transcriptase and terminal deoxynucleotidyl transferase.
• 2 ',3 '-O-Isopropylidenecytidine 5 -triphosphate inhibited DNA synthesis by reverse transcriptase and DNA polymerase beta, with its moiety incorporated in place of dTMP.
• Riboanhydroadenosine 5 -triphosphate demonstrated effective termination for all studied DNA polymerases.

Conclusions:

• Nucleotide analogues with restricted carbohydrate conformations can act as effective DNA synthesis termination substrates.
• Riboanhydroadenosine 5 -triphosphate is a versatile termination substrate for diverse DNA polymerases.
• This study pioneers the investigation of conformationally restricted analogues for DNA polymerase inhibition and termination.