DNA synthesis in vitro with an endoplasmic‐reticulum‐DNA‐polymerase complex from unfertilized sea urchin eggs

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Abstract
An endoplasmic-reticulum - DNA-polymerase complex was prepared from unfertilized sea urchin eggs and its DNA-synthesizing activity was examined using single-stranded DNA of bacteriophage fd as a template. The complex catalyzed the ribonucleotide-dependent DNA synthesis which required dNTPs, NTPs, Mg2+ and single-stranded DNA. The DNA synthesis was sensitive to aphidicolin and N-ethylmaleimide but was resistent to 2'',3''-dideoxyribosylthymine 5''-triphosphate (ddTTP) and .alpha.-amanitin, suggesting the involvement of DNA polymerase .alpha.. In parallel with the DNA synthesis, a small amount of RNA was synthesized in the presence of 100 .mu.g/ml .alpha.-amanitin. The Km value of ribonucleotides for the RNA synthesis coincided with that for the DNA synthesis, suggesting a correlation between the DNA and RNA syntheses. Labelling of the products with [.gamma.-32P]ATP followed by DNA digestion with pancreatic DNase I revealed the attachment of an oligoribonucleotide (7-11 bases in length) at the 5'' ends of the DNA products. These observations suggest that in DNA synthesis, primer RNA synthesis occurs first, followed by DNA chain elongation. During 1-90-min incubation, the amount of the DNA synthesized increased but the length was not significantly increased. Over 80% of the number of synthesized DNA molecules comprised a single population of short DNA fragments (60-200 bases, on average 120 bases in length) and the number of fragments increased, depending on the incubation time. However, DNA fragments of various sizes (about 100-6000 gases) were synthesized with DNA polymerase .alpha. solubilized from the endoplasmic-reticulum - DNA-polymerase complex. All this evidence suggests that in vitro, the complex preferentially synthesizes a particular size of short DNA fragments. The significance of the fragments is discussed.