The Effects of Cytosine Arabinoside on RNA-primed DNA Synthesis by DNA Polymerase α-Primase

Cindy Harrington,Fred W Perrino

doi:10.1074/jbc.270.44.26664

Abstract

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.

Highlights

In cells, RNA-primed DNA fragments are synthesized at origins of replication and on lagging strands at replication forks [1, 2]
Our results demonstrate that primase incorporates araCMP opposite dGMP as the 3Јterminal nucleotide of a full-length RNA primer very efficiently and that pol ␣ elongates the araCMP-terminated RNA primer
These data suggest that araCMP might be incorporated efficiently into RNA-primed DNA fragments at certain positions during discontinuous DNA synthesis in cells

Summary

EXPERIMENTAL PROCEDURES

The 10-mer RNA was from Oligo’s Etc. pol ␣-primase was purified [17], and unit definitions were as described [17, 18]. Primase reactions contained 100 pmol of 40-mer template, 6.3 units of primase, and 100 ␮M NTPs with [␣-32P]ATP. The pol ␣ reactions contained 0.25 pmol of 32P-5Ј-labeled primer hybridized to the 40-base DNA templates. Primase-coupled pol ␣ reactions contained 100 pmol of 40-mer template, 3.8 units of primase, 1.8 units of pol ␣, 100 ␮M NTPs, and 100 ␮M dCTP, dGTP, TTP with 10 ␮M [␣-32P]dATP. Primase and pol ␣ reactions were subjected to electrophoresis through 20% urea-polyacrylamide gels. Primase-coupled pol ␣ reactions were processed for scintillation spectroscopy by collecting acid-insoluble products on glass fiber disks

RESULTS

DNA araCMP dCMP

DISCUSSION