Heterogeneity of patch size in repair replicated DNA in Escherichia coli

Abstract

Repair replication in ultraviolet-irradiated Escherichia coli has been detected by the use of [ 3H]bromouracil as a density label and then equilibrium sedimentation in isopycnic CsCl gradients to separate the repaired parental DNA from normally replicated DNA. In the use of this assay it has been generally assumed that the patches of repair synthesis are short enough that the incorporated [ 3H]bromouracil will cause little detectable shift in density of the repaired DNA fragments. However, the repair label is consistently found to be skewed to the denser side of the parental DNA density position, possibly indicating size heterogeneity of the patches. We have now demonstrated such a heterogeneity. Repaired DNA isolated from the parental density regions of neutral CsCl gradients was extensively sheared by sonication to a single-strand molecular weight of 0.5 to 0.8 × 10 6 daltons. When the resulting fragments were sedimented in an alkaline CsCl gradient, a large fraction of the repair label was found in fragments which banded at density position sintermediate between those of fully bromouracil-labeled and unlabeled strands. Evidence is presented that this density-shift of some sonic fragments is due to the fact that repair replication sometimes produces very large patches. Base-composition analysis of the repaired regions indicates that these large patches must be at least as long as the sonic fragments (i.e. 1500 nucleotides). The possibility that the large patches arise from normal replication is eliminated. A model involving two alternative enzymic repair systems in the excision-repair process is presented to account for the existence of heterogeneity of patch size in repair replication.