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J. Biol. Chem., Vol. 278, Issue 23, 21276-21285, June 6, 2003

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Architecture of the Replication Complex and DNA Loops at the Fork Generated by the Bacteriophage T4 Proteins^*

Paul D. Chastain, II , Alexander M. Makhov , Nancy G. Nossal  and Jack Griffith  ¶

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

Rolling circle replication has previously been reconstituted in vitro using M13 duplex circles containing preformed forks and the 10 purified T4 bacteriophage replication proteins. Leading and lagging strand synthesis in these reactions is coupled and the size of the Okazaki fragments produced is typical of those generated in T4 infections. In this study the structure of the DNAs and DNA-protein complexes engaged in these in vitro reactions has been examined by electron microscopy. Following deproteinization, circular duplex templates with linear tails as great as 100 kb are observed. The tails are fully duplex except for one to three single-stranded DNA segments close to the fork. This pattern reflects Okazaki fragments stopped at different stages in their synthesis. Examination of the DNA-protein complexes in these reactions reveals M13 duplex circles in which 64% contain a single large protein mass (replication complex) and a linear duplex tail. In 56% of the replicating molecules with a tail there is at least one fully duplex loop at the replication complex resulting from the portion of the lagging strand engaged in Okazaki fragment synthesis folding back to the replisome. The single-stranded DNA segments at the fork bound by gene 32 and 59 proteins are not extended but rather appear organized into highly compact structures ("bobbins"). These bobbins constitute a major portion of the mass of the full replication complex.

Received for publication, February 13, 2003

^* This work was supported by grants from the National Institutes of Health NIDDK intramural program (to N. G. N.), the National Institutes of Health (to J. D. G.), and National Institutes of Health Grant GM31819. 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.

^¶ To whom correspondence should be addressed. Tel.: 919-966-2151; Fax: 919-966-3015; E-mail: jdg{at}med.unc.edu.

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