Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Summary

We present a procedure by which two-dimensional agarose-gel analysis can be used to identify the structure of replication intermediates that occur following UV irradiation.

Abstract

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.

Protocol

1. Growth and UV Irradiation.

1. 200μl of a fresh overnight culture containing the plasmid pBR322 grown in Davis medium¹ supplemented with 0.4% glucose, 0.2% casamino acids, and 10 μg/ml thymine (DGCthy medium) and 100 μg/ml ampicillin is pelleted. The cell pellet is then resuspended in 200μl DGCthy medium lacking ampicillin and used to inoculate 20 ml of DGCthy medium.
2. Cultures are grown without ampicillin selection in a shaking incubator at 37° C to an OD₆₀₀ of 0.5 (~ 5 x 10⁸ cells/ml). Growth without ampicillin avoids selection against abnormal or unproductive replication intermediates that may arise in some mu....

Discussion

Typical results obtained from wild type cells in the presence and absence of UV-induced damage are shown in Figure 1. In the absence of damage, ~1% of the total plasmid DNA can be found in the Y arc when cells are rapidly growing in exponential phase. Following irradiation, a transient increase in Y shaped molecules is observed as blocked replication forks accumulate at damaged sites. The X-shaped replication intermediates also transiently accumulate and persist until a time that correlates with when the lesions are rep.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
An example of the Southern analysis of the 2D gel probed	GE Healthcare	G15T8	Yellow Lighting
15 μwatt germicidal lamp	Sylvania	F20T12/GO	UV Lamp
Blak-Ray UV Intensity Meter 254nm	Daigger	EF28195T	UVC photometer
0.025 μm pore disks	Whatman, GE Healthcare	VSWP04700	Floating dialysis disks
PvuII	Fermentas	ER0632	Restriction Endonuclease
Nick-translation kit	Roche Group	976776	To make 32P-labeled probe
Blotting Paper	Whatman, GE Healthcare	3030-704	For Southern transfer
Nylon membrane	GE Healthcare	RPN203S	For Southern transfer