Chemical synthesis of an octanucleotide complementary to a portion of the cohesive end of P2 DNA and studies on the stability of duplex formation with P2 DNA.
A pyrimidine octanucleotide complementary to one of the cohesive ends of P2 DNA was chemically synthesized. Its sequence, d(C-T-T-T-C-C-C-C-OH), was verified by labeling it at the 5' end, followed by partial enzyme digestion and separation by a two-dimensional fingerprinting system. A single ribo-G residue was added to its 3' end using calf thymus deoxynucleotidyl terminal transferase. The resulting nonanucleotide primer was used in a detailed study on the stability of the duplexes formed in the partial as well as complete repair synthesis catalyzed by DNA polymerase I, at 5 degrees C in the presence of 70 mM potassium phosphate and 70 mM NaCl. The nonanucleotide primer was able to form a stable duplex with P2 DNA template only in the presence of DNA polymerase I. When the chain lengths of pyrimidine oligonucleotides were varied from 4 to 8 to test their abilities to serve as primers for the enzymatic repair synthesis, it was revealed that the minimum length required for the primer function is 8. Using the nonanucleotide as the primer and the right-hand cohesive end of the DNA as the template, repair synthesis was initiated simultaneously at the 3' end of the primer as well as at the right-hand 3' end of the DNA. This resulted in a decrease in the efficiency of repair synthesis at the 3' end of the primer, possibly due to the displacement of the primer by the enzyme. The enzyme was unable to displace the primer, when the primer was extended to a 13-mer prior to the initiation of repair synthesis at the 3'-OH end of the DNA. These data suggest that the strand displacement by DNA polymerase I at 5 degrees C in the presence of 70 mM potassium phosphate and 70 mM NaCl is not significant when the duplex is at least 13 nucleotides long. The efficiency of the repair synthesis at the 3'-OH end of the DNA-primer duplex could be increased by blocking the repair synthesis at the 3'-OH end of the DNA by converting it to 3'- phosphate. This method could be useful in DNA sequence analysis, where such specific repair synthesis is desired.
D001483:Base Sequence; D003090:Coliphages; D004256:DNA Polymerase I; D004260:DNA Repair; D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis*; D009841:Oligonucleotides / Q000138:chemical synthesis*; D013698:Templates, Genetic
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D004256:DNA Polymerase I