Covalently bound ribonucleotides in crab d(A-T) polymer

Area of Science:

• Biochemistry
• Molecular Biology
• Marine Biology

Background:

• The DNA polymer d(A-T)n from Cancer borealis is known to contain 3% guanine + cytosine (G+C) residues.
• Previous analysis did not account for the presence of ribonucleotides within this DNA polymer.

Purpose of the Study:

• To investigate the presence and nature of ribonucleotides within purified crab d(A-T) polymer.
• To determine the distribution and linkage of these ribonucleotides within the DNA structure.

Main Methods:

• Digestion of d(A-T)n with crude venom followed by high-pressure liquid chromatography (HPLC) analysis of nucleosides.
• Exhaustive digestion with pancreatic DNase I to generate dinucleotides, followed by HPLC fractionation.
• Hydrolysis with potassium hydroxide (KOH) at 37°C to analyze ribomononucleotides and deoxyribomononucleotides.

Main Results:

• Up to 50% of guanosine was identified as riboguanosine (rG) after venom digestion.
• Three mixed deoxyribo-ribonucleotide dinucleotides (-dC-rG, -dT-rA, -dT-rG) were detected after DNase I digestion.
• KOH hydrolysis revealed dispersed ribonucleotides, with some linked to longer deoxyribonucleotide fragments, suggesting 3'(2')-terminal attachment.

Conclusions:

• Crab d(A-T) polymer from C. borealis contains 1-3% dispersed, covalently bound ribonucleotides.
• These ribonucleotides are integrated within the DNA structure, not merely contaminants.
• DNase I's sugar specificity may be influenced by the nucleotide preceding the cleavage site.