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Unidirectional replication as visualized by two-dimensional agarose gel electrophoresis.

Abstract：

:Two-dimensional (2D) agarose gel electrophoresis is progressively replacing electron microscopy as the technique of choice to map the initiation and termination sites for DNA replication. Two different versions were originally developed to analyze the replication of the yeast 2 microns plasmid. Neutral/Neutral (N/N) 2D agarose gel electrophoresis has subsequently been used to study the replication of other eukaryotic plasmids, viruses and chromosomal DNAs. In some cases, however, the results do not conform to the expected 2D gel patterns. In order to better understand this technique, we employed it to study the replication of the colE1-like plasmid, pBR322. This was the first time replicative intermediates from a unidirectionally replicated plasmid have been analyzed by means of N/N 2D agarose gel electrophoresis. The patterns obtained were significantly different from those obtained in the case of bidirectional replication. We showed that identification of a complete are corresponding to molecules containing an internal bubble is not sufficient to distinguish a symmetrically located bidirectional origin from an asymmetrically located unidirectional origin. We also showed that unidirectionally replicated fragments containing a stalled fork can produce a pattern with an inflection point. Finally, replication appeared to initiate at only some of the potential origins in each multimer of pBR322 DNA.

journal_name

J Mol Biol

journal_title

Journal of molecular biology

authors

Martín-Parras L,Hernández P,Martínez-Robles ML,Schvartzman JB

doi

10.1016/0022-2836(91)90357-c

subject

Has Abstract

pub_date

1991-08-20 00:00:00

pages

843-53

issue

4

eissn

0022-2836

issn

1089-8638

pii

0022-2836(91)90357-C

journal_volume

220

pub_type

杂志文章

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