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J. Biol. Chem., Vol. 281, Issue 32, 22635-22646, August 11, 2006

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The Replication Intermediates in Escherichia coli Are Not the Product of DNA Processing or Uracil Excision^*

From the Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801-3709

The current model of DNA replication in Escherichia coli postulates continuous synthesis of the leading strand, based on in vitro experiments with purified enzymes. In contrast, in vivo experiments in E. coli and its bacteriophages, in which maturation of replication intermediates was blocked, report discontinuous DNA synthesis of both the lagging and the leading strands. To address this discrepancy, we analyzed nascent DNA species from ThyA⁺ E. coli cells replicating their DNA in ligase-deficient conditions to block maturation of replication intermediates. We report here that the bulk of the newly synthesized DNA isolated from ligase-deficient cells have a length between 0.3 and 3 kb, with a minor fraction being longer that 11 kb but shorter than the chromosome. The low molecular weight of the replication intermediates is unchanged by blocking linear DNA processing with a recBCD mutation or by blocking uracil excision with an ung mutation. These results are consistent with the previously proposed discontinuous replication of the leading strand in E. coli.

Received for publication, March 13, 2006 , and in revised form, June 8, 2006.

^* This work was supported by National Institutes of Health (NIH) Grant T32 GM07283, NIH Grant F31 GM075425 (to L. A.), American Cancer Society Grant RSG-05-135-01-GMC, and startup funds from the University of Illinois. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Microbiology, University of Illinois at Urbana-Champaign, B103 C&LSL, 601 S. Goodwin Ave., Urbana, IL 61801-3709. Tel.: 217-265-0329; Fax: 217-244-6697; E-mail: kuzminov{at}life.uiuc.edu.

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