Effects of Arabinosylcytosine-Substituted DNA on DNA/RNA Hybrid Stability and Transcription by T7 RNA Polymerase

Abstract

Cytosine arabinoside (araC) is a potent antileukemic agent which interferes with DNA replication both as a dNTP competitive inhibitor as well as after its misincorporation into DNA. We previously developed a chemical methodology for the synthesis of DNA oligomers containing araC which allowed us to study its site specific effects on duplex stability and chemical reactivity [Beardsley, G. P., Mikita, T., Klaus, M., & Nussbaum, A. (1988) Nucleic Acids Res. 16, 9165], as well as its effects on DNA ligase and DNA polymerase activity [Mikita, T., & Beardsley, G. P. (1988) Biochemistry 27, 4698]. The DNA polymerase studies, in addition to other observations, showed that araC in DNA templates could have an inhibitory effect on polymerase bypass. As a template lesion, there exists the potential for interference with other aspects of DNA metabolism, such as transcription. We have characterized a DNA/RNA hybrid containing an araC-G base pair, comparing thermal stability, chemical cleavage rates, and duplex gel mobility to an identically sequenced DNA duplex. We find that the A-form DNA/RNA hybrid and the B-form DNA duplex are nearly identical in the extent their thermal stability is affected by an araC-G(dG) base pair. Substitutions of araC for dC were made at various positions in a series of DNA duplex substrates containing a T7 RNA polymerase promoter with variable length coding strands. These were used to probe the effect of araC on promoter recognition, initiation, and elongation by T7 RNA polymerase in vitro. Substitutions in the central promoter region had no observable effect on RNA polymerase binding, initiation rate, or transcriptional output. Coding strand substitutions defined an area of high sensitivity in the initiation region where miss-starts, primer slippage, and an inability to escape from abortive cycling occur depending on the position substituted. Substitutions after position 10 had little effect on transcription output. These highly variable, position dependent effects indicate a narrow window of vulnerability where transcription output is severely reduced (approximately 100-fold) by a subtle DNA lesion that has little or no consequence when situated elsewhere in these small coding units.

The Antileukemia Drug 2-Chloro-2′-deoxyadenosine: An Intrinsic Transcriptional Antagonist

“…Cytosine arabinoside substitutions in the central promoter region for phage T7 RNA polymerase did not modify polymerase binding, initiation, or transcriptional output (Mikita and Beardsley, 1994). However, phage RNA polymerases do not rely on the formation of a multiprotein initiation complex involving TBP but rather are sequence-dependent.…”

Transcription Arrest at a Lesion in the Transcribed DNA Strand in Vitro Is Not Affected by a Nearby Lesion in the Opposite Strand

“…Previous studies have investigated the effect of several types of lesions on transcription elongation by T7 RNAP. Psoralen monoadducts and diadducts (25), benzo(a)pyrene (26,27), benzo(c)phenanthrene (28), tetrahydrofuran, acetylaminofluorene, and aminofluorene (29), and thymine glycol (30,31) block the elongating T7 RNAP when present in the transcribed strand, whereas aminofluorene (29) and cytosine arabinose do not block it (32).…”

“…Initially, preinitiation complexes were formed by incubating rat liver protein fractions D (2 g, containing TFIID and TFIIH), B (1 g, TFIIF/TFIIE), recombinant rat TFIIB (3 ng), and rat liver RNAP II (0.5 g) at 28°C for 30 min. 20 M ATP and UTP and 40 Ci of [␣- 32 P]CTP (800 Ci/mmol) were added to the reaction for the labeling of nascent transcripts. Transcription was synchronized as all transcribing polymerases paused at position 15, at which the first GTP was required for further elongation.…”

A model for DNA polymerase translocation: worm‐like movement of DNA within the binding cleft

“…The A-like form is preferable for RNA-DNA hybrid duplexes [32,33] (and for some short duplexes which contains the ribonucleotide insertion [34]), hence the reverse B ~ A transition within the binding channel should have a lower energetic barrier in these cases. Mikita and Beardsley [35,36] have shown that DNA replication can be arrested by structural lesion of arabinosylcytosine in. the template DNA strand.…”

“…Structural studies on Bform DNA all indicate that the perturbations introduced by araC are slight to moderate in their effect, particularly compared to those changes introduced by many of the more bulky modifications of the DNA bases which grossly disturb duplex structure and stability. However, its inhibitory effects on DNA polymerase can be equal to or greater than some of these more aggressive structural lesions [36]. On the other hand, a molecular modeling study showed that strong steric clashes occurred when araC-G base pairs were built into an A-form helix environment [37].…”