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J Biol Chem, Vol. 273, Issue 50, 33386-33396, December 11, 1998

DnaB Helicase Is Unable to Dissociate RNA-DNA Hybrids
ITS IMPLICATION IN THE POLAR PAUSING OF REPLICATION FORKS AT ColE1 ORIGINS

David Santamaría, Guillermo de la Cueva^§, María Luisa Martínez-Robles, Dora B. Krimer, Pablo Hernández, and Jorge B. Schvartzman

From the  Departamento de Biología Celular y del Desarrollo and ^§ Departamento de Microbiología Molecular, CIB (Consejo Superior de Investigaciones Científicas), Velázquez 144, 28006 Madrid, Spain

A series of plasmids were constructed containing two unidirectional ColE1 replication origins in either the same or opposite orientations and their replication mode was investigated using two-dimensional agarose gel electrophoresis. The results obtained showed that, in these plasmids, initiation of DNA replication occurred at only one of the two potential origins per replication round regardless of origins orientation. In those plasmids with inversely oriented origins, the silent origin act as a polar pausing site for the replication fork initiated at the other origin. The distance between origins (up to 5.8 kilobase pairs) affected neither the interference between them to initiate replication nor the pausing function of the silent origin. A deletion analysis indicated that the presence of a transcription promoter upstream of the origin was the only essential requirement for it to initiate replication as well as to account for its polar pausing function. Finally, in vitro helicase assays showed that Escherichia coli DnaB is able to melt DNA-DNA homoduplexes but is very inefficient to unwind RNA-DNA hybrids. Altogether, these observations strongly suggest that replication forks pause at silent ColE1 origins due to the inability of DnaB helicase, which leads the replication fork in vivo, to unwind RNA-DNA hybrids.

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