THE EFFECTS OF CYTOSINE-ARABINOSIDE OIL RNA-PRIMED DNA-SYNTHESIS BY DNA-POLYMERASE ALPHA-PRIMASE

Oligonucleotides containing a specific initiation site for polymerase alpha-primase (pol alpha-primase) were used to measure the effects of cytosine arabinoside triphosphate and cytosine arabinoside monophosphate (araCMP) in DNA on RNA-primed DNA synthesis. Primase inserts araCMP at the 3' terminus of a full-length RNA primer with a 400-fold preference over GRIP. The araCMP is elongated efficiently by pol alpha in the primase-coupled reaction. Extension from RNA 3'-araCMP is 50-fold less efficient than from CMP, and extension from DNA 3'-araCMP is 1600-fold less efficient than from dCMP. Using araCMP-containing templates, primer synthesis is reduced 2-3 fold, and RNA-primed DNA synthesis is reduced 2-8-fold. The efficiency of polymerization past a template araCMP by pol alpha is reduced 180-fold during insertion of dGMP opposite araCMP and 35-fold during extension from the araCMP:dGMP 3' terminus. These results show that the pol alpha-primase efficiently incorporates araCMP as the border nucleotide between RNA and DNA and suggest that the inhibitory effects of araC most likely result from slowed elongation of pol a: and less so from inhibition of primer synthesis by primase.