HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 282, Issue 2, 1098-1108, January 12, 2007

This Article Full Text Full Text (PDF) All Versions of this Article: 282/2/1098    most recent M606772200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Nossal, N. G. Articles by Griffith, J. D. Search for Related Content PubMed PubMed Citation Articles by Nossal, N. G. Articles by Griffith, J. D. Social Bookmarking                      What's this?

Architecture of the Bacteriophage T4 Replication Complex Revealed with Nanoscale Biopointers^*

Nancy G. Nossal, Alexander M. Makhov, Paul D. Chastain, II, Charles E. Jones, and Jack D. Griffith¹

From the Laboratory of Molecular and Cellular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0830 and the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7295

Our previous electron microscopy of DNA replicated by the bacteriophage T4 proteins showed a single complex at the fork, thought to contain the leading and lagging strand proteins, as well as the protein-covered single-stranded DNA on the lagging strand folded into a compact structure. "Trombone" loops formed from nascent lagging strand fragments were present on a majority of the replicating molecules (Chastain, P., Makhov, A. M., Nossal, N. G., and Griffith, J. D. (2003) J. Biol. Chem. 278, 21276–21285). Here we probe the composition of this replication complex using nanoscale DNA biopointers to show the location of biotin-tagged replication proteins. We find that a large fraction of the molecules with a trombone loop had two pointers to polymerase, providing strong evidence that the leading and lagging strand polymerases are together in the replication complex. 6% of the molecules had two loops, and 31% of these had three pointers to biotin-tagged polymerase, suggesting that the two loops result from two fragments that are being extended simultaneously. Under fixation conditions that extend the lagging strand, occasional molecules show two nascent lagging strand fragments, each being elongated by a biotin-tagged polymerase. T4 41 helicase is present in the complex on a large fraction of actively replicating molecules but on a smaller fraction of molecules with a stalled polymerase. Unexpectedly, we found that 59 helicase-loading protein remains on the fork after loading the helicase and is present on molecules with extensive replication.

Received for publication, July 17, 2006 , and in revised form, November 13, 2006.

The co-authors all wish to dedicate this work to our first author, Dr. Nancy Nossal, who sadly passed away following the completion of this work. In the finest tradition of first authorship she carried out the greatest share of the experimental work in this report, including crafting the final product.

^* This work was supported by the Intramural Research Program of NIDDK, National Institutes of Health (NIH) (to N. G. N.) and by NIH Grant GM31819 (to J. D. G.). 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: Lineberger Comprehensive Cancer Center, University of North Carolina, Mason Farm Road, Chapel Hill, NC 27599-7295. Tel.: 919-966-2151; Fax: 919-66-3015; E-mail: jdg{at}med.unc.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?