Abstract
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.
Highlights
DNA replication is accomplished by a highly organized DNAprotein ensemble, operating as a molecular machine that replicates both strands in a highly coordinated manner
Evidence for the coordination of leading and lagging strand synthesis obtained in E. coli [5, 8, 9], T4 [2, 10, 11], and T7 [12] was based on gel electrophoretic demonstrations that the average length distribution of Okazaki fragments did not change upon dilution of the polymerase or the helicase/primase
In this paper we utilized the highly purified T4 replication proteins to probe the architecture of the DNA strands and protein complex at a rolling circle replication fork
Summary
Vol 278, No 23, Issue of June 6, pp. 21276 –21285, 2003 Printed in U.S.A. Architecture of the Replication Complex and DNA Loops at the Fork Generated by the Bacteriophage T4 Proteins*. In the T7 studies, the single-stranded DNA (ssDNA) segments at the fork were not extended as predicted in the original models but rather were in compact bodies that were termed “bobbins.” This may reflect the weaker binding of the T7 gene 2.5 ss-binding protein as contrasted to the robust binding of the T4 gene 32 or E. coli SSB proteins to ssDNA, or may represent a general feature of DNA replication machines. Bacteriophage T4 provides a natural extension with which to examine the generality of the findings made with T7 It is replicated by 10 different proteins including a sliding clamp and clamp loading system typical of E. coli and complex eukaryotic systems; each of the 10 proteins has been extensively purified and studied We describe the first visualization of a trombone loop in the T4 system and the finding that the ssDNA segments involved in Okazaki fragment synthesis are organized into compact “bobbins.”
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