Stereochemical control of DNA biosynthesis

Area of Science:

• Biochemistry
• Molecular Biology
• Nucleic Acid Chemistry

Background:

• DNA biosynthesis relies on precise stereochemical control.
• The incorporation of non-natural L-nucleotides into DNA can reveal insights into enzyme mechanisms.
• Understanding DNA polymerase fidelity is crucial for genetic stability and therapeutic applications.

Purpose of the Study:

• To investigate the stereochemical control during DNA biosynthesis when L-nucleotide residues are incorporated.
• To compare the selectivity of different DNA polymerases towards L-nucleotides.
• To analyze the impact of L-nucleotide incorporation on DNA template-primer complex geometry.

Main Methods:

• Enzymatic synthesis using DNA-synthesizing complexes with single L-nucleotide substitutions.
• Testing of template-dependent DNA polymerases (E. coli DNA polymerase I Klenow fragment, Thermus aquaticus DNA polymerase, avian myeloblastosis virus reverse transcriptase) and template-independent calf-thymus terminal deoxynucleotidyl transferase.
• Analysis of substrate utilization (L-dNTPs) and product formation (dinucleoside tetraphosphates).
• Geometric modeling of template-primer complexes.

Main Results:

• Template-dependent DNA polymerases demonstrated stringent stereoselectivity against L-nucleotide incorporation.
• Calf-thymus terminal deoxynucleotidyl transferase showed lower selectivity.
• DNA polymerase I and reverse transcriptase formed dinucleoside 5',5'-tetraphosphates with L-dTTP.
• Incorporation of a single L-nucleotide residue significantly altered template-primer complex geometry.

Conclusions:

• Template-dependent DNA polymerases possess robust mechanisms for stereochemical fidelity during DNA synthesis.
• Terminal deoxynucleotidyl transferase exhibits a relaxed stereochemical preference.
• The geometric constraints imposed by L-nucleotide incorporation highlight the precise nature of DNA polymerase active sites.