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Action of intact AP (apurinic/apyrimidinic) sites and AP sites associated with breaks on the transcription of T7 coliphage DNA by Escherichia coli RNA polymerase.
Biochem J. 1985 Jul 01; 229(1):173-81.BJ

Abstract

The effect of apurinic/apyrimidinic (AP) sites in DNA on RNA and protein synthesis was studied in vitro using T7 coliphage DNA. Initiation of RNA synthesis by Escherichia coli RNA polymerase was synchronized and heparin was used to prevent reinitiation. When the T7 DNA contained AP sites, the rate of RNA synthesis was decreased but it remained higher than the values calculated on the assumption that an AP site in the transcribed strand is a complete block to the enzyme progression. Moreover, after the time taken by an unimpeded enzyme to go from promoter to terminator, the rate of RNA synthesis remained elevated and the number of complete RNA molecules (7000 nucleotides) continued to increase for some time. These results suggest that, if the E. coli RNA polymerase is stopped by an AP site, most often, after a pause, the enzyme resumes elongation of the RNA chain which is continuous over the AP site. Sometimes however, RNA synthesis is definitively interrupted during the pause; the probability of interruption has been estimated to be 0.3 in our experimental conditions. When a nick is placed 5' to the AP site by an AP endonuclease, the results are similar: most often, the RNA chain is synthesized without interruption past the nick in the template strand. The pause of the E. coli RNA polymerase at this combined lesion appears to be shorter than when the AP site is intact. To investigate whether a nucleotide is placed in the RNA chain in front of the AP site in the template strand by E. coli RNA polymerase, RNA synthesis was taken to completion before using this RNA for protein synthesis and measuring the activity of gene-1 product, T7 RNA polymerase. The result suggests that, after pausing, the E. coli RNA polymerase places a nucleotide in the RNA chain when passing over an AP site. The mechanism of the delayed lethality of T7 coliphages treated with monofunctional alkylating agents, which is due to the appearance of AP sites, is discussed.

Authors

No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

2412545

Citation

Flamée, P A., and W G. Verly. "Action of Intact AP (apurinic/apyrimidinic) Sites and AP Sites Associated With Breaks On the Transcription of T7 Coliphage DNA By Escherichia Coli RNA Polymerase." The Biochemical Journal, vol. 229, no. 1, 1985, pp. 173-81.
Flamée PA, Verly WG. Action of intact AP (apurinic/apyrimidinic) sites and AP sites associated with breaks on the transcription of T7 coliphage DNA by Escherichia coli RNA polymerase. Biochem J. 1985;229(1):173-81.
Flamée, P. A., & Verly, W. G. (1985). Action of intact AP (apurinic/apyrimidinic) sites and AP sites associated with breaks on the transcription of T7 coliphage DNA by Escherichia coli RNA polymerase. The Biochemical Journal, 229(1), 173-81.
Flamée PA, Verly WG. Action of Intact AP (apurinic/apyrimidinic) Sites and AP Sites Associated With Breaks On the Transcription of T7 Coliphage DNA By Escherichia Coli RNA Polymerase. Biochem J. 1985 Jul 1;229(1):173-81. PubMed PMID: 2412545.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Action of intact AP (apurinic/apyrimidinic) sites and AP sites associated with breaks on the transcription of T7 coliphage DNA by Escherichia coli RNA polymerase. AU - Flamée,P A, AU - Verly,W G, PY - 1985/7/1/pubmed PY - 1985/7/1/medline PY - 1985/7/1/entrez SP - 173 EP - 81 JF - The Biochemical journal JO - Biochem. J. VL - 229 IS - 1 N2 - The effect of apurinic/apyrimidinic (AP) sites in DNA on RNA and protein synthesis was studied in vitro using T7 coliphage DNA. Initiation of RNA synthesis by Escherichia coli RNA polymerase was synchronized and heparin was used to prevent reinitiation. When the T7 DNA contained AP sites, the rate of RNA synthesis was decreased but it remained higher than the values calculated on the assumption that an AP site in the transcribed strand is a complete block to the enzyme progression. Moreover, after the time taken by an unimpeded enzyme to go from promoter to terminator, the rate of RNA synthesis remained elevated and the number of complete RNA molecules (7000 nucleotides) continued to increase for some time. These results suggest that, if the E. coli RNA polymerase is stopped by an AP site, most often, after a pause, the enzyme resumes elongation of the RNA chain which is continuous over the AP site. Sometimes however, RNA synthesis is definitively interrupted during the pause; the probability of interruption has been estimated to be 0.3 in our experimental conditions. When a nick is placed 5' to the AP site by an AP endonuclease, the results are similar: most often, the RNA chain is synthesized without interruption past the nick in the template strand. The pause of the E. coli RNA polymerase at this combined lesion appears to be shorter than when the AP site is intact. To investigate whether a nucleotide is placed in the RNA chain in front of the AP site in the template strand by E. coli RNA polymerase, RNA synthesis was taken to completion before using this RNA for protein synthesis and measuring the activity of gene-1 product, T7 RNA polymerase. The result suggests that, after pausing, the E. coli RNA polymerase places a nucleotide in the RNA chain when passing over an AP site. The mechanism of the delayed lethality of T7 coliphages treated with monofunctional alkylating agents, which is due to the appearance of AP sites, is discussed. SN - 0264-6021 UR - https://www.unboundmedicine.com/medline/citation/2412545/Action_of_intact_AP__apurinic/apyrimidinic__sites_and_AP_sites_associated_with_breaks_on_the_transcription_of_T7_coliphage_DNA_by_Escherichia_coli_RNA_polymerase_ L2 - https://portlandpress.com/biochemj/article-lookup/doi/10.1042/bj2290173 DB - PRIME DP - Unbound Medicine ER -