POST‐INCISION EVENTS IN EXCISION REPAIR IN ESCHERICHIA COLI

The removal from DNA of UV-induced pyrimidine dimers, as well as a variety of chemically-induced lesions, is accomplished by a complex enzymatic process called excision repair. While the main features of excision repair are well known, its genetic and enzymatic details are poorly understood. In essence, the process formally consists of four steps. In the first, a single-stranded nick (or incision) is made adjacent to the dimer. The incision step is complex and requires ATP and the function of at least two genes, uurA and uurB. A mutation at either locus will completely prevent dimer excision [10,24]. In the second step a small segment of DNA containing the dimer is digested in the 5’ + 3’ direction (excision). Thirdly, the digested material is resynthesized (repair replication). Resynthesis and excision are difficult to separate experimentally and it is not clear which actually precedes the other. No single mutation has been described that entirely blocks excision repair because it prevents either of these steps. The excision and resynthesis steps appear to be organized into two distinct modes or pathways. One produces short repair patches (less than 30 nucleotides) and the other produces long patches (several hundred nucleotides or more; [ 111). There thus appear to be multiple, redundant enzymatic activities able to carry out these steps and to fill in for one another if need be. In the final step, ligation, the newly synthesized repair patch is covalently bound to the parental DNA. Each step in excision repair appears to be closely coordinated with the other steps. Shortly after irradiation transient incision breaks can be detected by alkaline sucrose density gradient sedimentation but the maximum number of breaks, usually 8-10, is the same regardless of the dose and is far fewer than the number of dimers. What is dose-dependent is the time during which breaks can be detected. There thus appears to be about 10 breaks made initially with no new incisions being made until the first ones are closed. The result is the appearance of a constant low number of breaks during the repair period but a different population of breaks open at any given time [54]. The manner in which all of these steps is coordinated is not known. The purpose of this article is to review recent literature dealing with post-incision events and to speculate on the proper sequence of steps, their genetic control, and the regulation of patch size. A number of techniques exist for measuring various aspects of excision repair. Thus one can measure the rate at which dimers are released from the DNA, the . rate at which incisions are made and joined, the amount of repair replication, and the length of the resulting repair patch. These have been done both in uiuo and in oitro using permeabilized cells. Further, assessments of excision repair can be made by measuring host-cell reactivation (HCR), the ability of unirradiated cells to promote the recovery of certain irradiated phages. HCR is mostly an excision repairdependent phenomenon although a minor component is dependent on other repair processes [47]. uurA and uurB mutants do not make incisions and are completely excision deficient as measured by the techniques above [10,24,58]. Mutations in the genes listed below allow incision but affect or have the potential of affecting post-incision events :