Effects of 2-chloro-2'-deoxyadenosine 5'-triphosphate on DNA synthesis in vitro by purified bacterial and viral DNA polymerase

Abstract

2-Chloro-2'-deoxyadenosine 5'-triphosphate (CldATP) was compared with dATP as a substrate for DNA synthesis by bacterial and viral DNA polymerases in vitro. Lengths of chain extension and DNA synthesis pause sites were determined by comparison with products generated by dideoxynucleotide sequencing methods on the same end-labeled primer/template duplex after high-resolution polyacrylamide gel electrophoresis. Reverse transcriptase (RT) from human immunodeficiency virus (HIV-1) and avian myeloblastosis virus (AMV) incorporated CldATP efficiently. DNA strand elongation continued past most chloroadenine (ClA) insertion sites but resulted in shorter chains than when dATP was inserted. Phage T4 DNA polymerase incorporated CldATP least efficiently; Klenow fragment of Escherichia coli DNA polymerase I and modified T7 DNA polymerase (Sequenase) showed intermediate ability to utilize the analogue. Incorporation of several consecutive ClA residues into the replicating strand dramatically reduced the ability of Sequenase, Klenow fragment, and T4 DNA polymerases to continue strand elongation. In the absence of the corresponding normal deoxyribonucleoside triphosphate during DNA synthesis, ClA was frequently misincorporated as thymine, cytosine, or guanine by both AMV RT and HIV-1 RT but rarely, if at all, by Klenow fragment, Sequenase, and T4 DNA polymerase. Except T4, for most DNA polymerases, CldATP at 10-20-fold molar excess over dATP was not a strong competitive inhibitor of dATP, as judged by the amount of strand extension and polymerase pause sites during DNA synthetic reactions. Our results indicate that the degree of strand extension in the presence of CldATP, the number and location of polymerase pause sites, and the amount of misincorporation of the analogue are both polymerase- and sequence-dependent.

Mechanisms of apoptosis induction by nucleoside analogs

“…It is modified only on the nucleobase and contains a normal 2 0 -deoxyribose carbohydrate moiety. Accordingly, the triphosphate readily incorporated into DNA, and thereafter is also a fair substrate for extension (Hentosh et al, 1990). As with fludarabine, this process may be facilitated by the inhibitory activity of the triphosphate against ribonucleotide reductase, which seems to be similar in mechanism, but more potent than that of fludarabine triphosphate (Parker et al, 1988).…”

A Comparison of the Transportability, and Its Role in Cytotoxicity, of Clofarabine, Cladribine, and Fludarabine by Recombinant Human Nucleoside Transporters Produced in Three Model Expression Systems

“…Once inside the cell, Cl-dAdo is phosphorylated by cytosolic deoxycytidine kinase (Carson et al, 1983) and mitochondrial deoxyguanosine kinase (Wang et al, 1993). The cytotoxicity of Cl-dAdo to proliferating cells is due to interruption of DNA synthesis by Cl-dATP inhibition of ribonucleotide reductase and DNA polymerases ␣ and ␤ with resultant dNTP pool imbalances and DNA strand breaks (Griffig et al, 1989;Hirota et al, 1989;Hentosh et al, 1991). The mechanism of cytotoxicity of Cl-dAdo to nondividing cells is by apoptosis (Carson et al, 1983;Seto et al, 1985;Robertson et al, 1993).…”

Hoogsteen vs. Watson–Crick Base Pairing: Incorporation of 2‐Substituted Adenine‐ and 7‐Deazaadenine 2′‐Deoxy‐β‐D‐ribonucleosides into Oligonucleotides

“…It was found that it is resistant toward adenosine deaminase and intracellularly phosphorylated to the 5'-monophosphate by cytidine kinase [27]. Subsequently, the 5'-monophosphate is phosphorylated to the 5'-triphosphate by cellular enzymes which is then incorporated into DNA of dividing cells [28] [29]. After five consecutive additions, DNA synthesis is virtually terminated [29] -an effect which probably makes a major contribution to the cytotoxicity of Cl 2 A d .…”