The Application of a Minicircle Substrate in the Study of the Coordinated T4 DNA Replication

Jingsong Yang, Michael A. Trakselis, Rosa Maria Roccasecca, Stephen Benkovic

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

A reconstituted in vitro bacteriophage T4 DNA replication system was studied on a synthetic 70-mer minicircle substrate. This substrate was designed so that dGMP and dCMP were exclusively incorporated into the leading and the lagging strand, respectively. This design allows the simultaneous and independent measurement of the leading and lagging strand synthesis. In this paper, we report our results on the characterization of the 70-mer minicircle substrate. We show here that the minicircle substrate supports coordinated leading and lagging strand synthesis under the experimental conditions employed. The rate of the leading strand fork movement was at an average of ∼150 nucleotides/s. This rate decreased to less than 30 nucleotides/s when the helicase was omitted from the reaction. These results suggest that both the holoenzyme and the primosome can be simultaneously assembled onto the minicircle substrate. The lagging strand synthesized on this substrate is of an average of 1.5 kb, and the length of the Okazaki fragments increased with decreasing [rNTPs]. The proper response of the Okazaki fragment size toward the change of the priming signal further indicates a functional replisome assembled on the minicircle template. The effects of various protein components on the leading and lagging strand synthesis were also studied. The collective results indicate that coordinated strand synthesis only takes place within certain protein concentration ranges. The optimal protein levels of the proteins that constitute the T4 replisome generally bracket the concentrations of the same proteins in vivo. Omission of the primase has little effect on the rate of dNMP incorporation or the rate of the fork movement on the leading strand within the first 30 s of the reaction. This inhibition only becomes significant at later times of the reaction and may be associated with the accumulation of single-stranded DNA leading to the collapse of active replisomes.

Original languageEnglish (US)
Pages (from-to)49828-49838
Number of pages11
JournalJournal of Biological Chemistry
Volume278
Issue number50
DOIs
StatePublished - Dec 12 2003

Fingerprint

DNA Replication
DNA
Substrates
Proteins
Nucleotides
DNA Primase
Bacteriophage T4
Holoenzymes
Single-Stranded DNA
Bacteriophages
Okazaki fragments

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Yang, Jingsong ; Trakselis, Michael A. ; Roccasecca, Rosa Maria ; Benkovic, Stephen. / The Application of a Minicircle Substrate in the Study of the Coordinated T4 DNA Replication. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 50. pp. 49828-49838.
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The Application of a Minicircle Substrate in the Study of the Coordinated T4 DNA Replication. / Yang, Jingsong; Trakselis, Michael A.; Roccasecca, Rosa Maria; Benkovic, Stephen.

In: Journal of Biological Chemistry, Vol. 278, No. 50, 12.12.2003, p. 49828-49838.

Research output: Contribution to journalArticle

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