The effects of cytosine arabinoside on 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 CMP. 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 alpha and less so from inhibition of primer synthesis by primase.