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

Abstract

Oligonucleotides containing a specific initiation site for polymerase ␣-primase (pol ␣-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 ␣ in the primasecoupled 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 ␣ 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 ␣-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 ␣ and less so from inhibition of primer synthesis by primase.In cells, RNA-primed DNA fragments are synthesized at origins of replication and on lagging strands at replication forks (1, 2). Initiation of DNA chains requires oligoribonucleotide synthesis to provide 3Ј-hydroxyl termini for DNA polymerase elongation. The tight association between DNA polymerase ␣ (pol ␣) 1 and primase indicate that pol ␣-primase complex is responsible for RNA-primed DNA synthesis. Primase synthesizes RNA primers that are about 10 Nts in length de novo, and pol ␣ elongates the primers.The aranucleotides inhibit DNA replication in animal cells (3). Inhibition by the triphosphate forms of these analogues might result during RNA-primed DNA synthesis by pol ␣-primase (4). Incorporation into RNA primers by primase and into DNA by pol ␣ is at positions opposite complementary nucleotides (3-10). Primase inserts araATP and FaraATP more efficiently than ATP (9, 11), and pol ␣ inserts these analogues as efficiently as deoxynucleotides (12). Insertion of aranucleotides terminates primer chain elongation by primase (9, 11), and 3Ј-aranucleotides in DNA decrease elongation by pol ␣ 2000-fold (12). In contrast, aranucleotides at RNA 3Ј termini only moderately decrease elongation by pol ␣ (9, 11). The synthesis of nonfunctional primers less than 7 Nts in length using homopolymer templates has made it difficult to quantitate effects of aranucleotides during RNA-primed DNA synthesis and to distinguish effects on primase and pol ␣.Oligonucleotide templates permit analysis of sequence-specific effects of aranucleotides on RNA-primed DNA synthesis. The sequences of some primer initiation sites have been identified (13-16), and these sequences support initiation in oligomer templates (15, 16). Using an oligonucleotide containing an initiation site for pol ␣-primase, primers with the sequence 5Ј-GGAAGAAAGC-3Ј are generated (16). Incorporation of araCMP at the 3Ј terminus...

DNA Polymerase β Ribonucleotide Discrimination

“…5B). Interestingly, similar results were observed for pol ␣ (B-family) (36,39). Thus, O3Ј of the rNMP-primer terminus must be well positioned for further catalysis.…”

Rescue of Mesencephalic Dopamine Neurons by Anticancer Drug Cytosine Arabinoside

“…That under our culture conditions, the astrocytes are proliferating and, therefore, actively replicating their DNA, might also explain their greater sensitivity to the antimitotic agent. In cancer cells, ara-C is thought to inhibit DNA polymerase activity after incorporation of ara-C triphosphate into DNA, blocking replication (Graham and Whitmore, 1970;Harrington and Perrino, 1995), without affecting its RNA primer (Harrington and Perrmno, 1995). This also appears to be its mode of action on the astrocytes in our cultures, where intense nuclear labeling with tritiated ara-C was accompanied by cessation of cell division.…”

Identification and Expression of the TREX1 and TREX2 cDNA Sequences Encoding Mammalian 3′→5′ Exonucleases

“…The 35-mer⅐21-mer duplexes contain one to eight mispaired nucleotides at the 3Ј terminus. The RNA 10-mer, araCMPterminated 21-mer, and the complementary DNA templates have been described (19,28). Enzyme dilutions were prepared in 1 mg/ml bovine serum albumin at 4°C.…”