PROCESSING OF MODEL SINGLE-STRAND BREAKS IN PHI-X-174 RF TRANSFECTING DNA BY ESCHERICHIA-COLI

被引:25
作者
KOW, YW [1 ]
FAUNDEZ, G [1 ]
MELAMEDE, RJ [1 ]
WALLACE, SS [1 ]
机构
[1] UNIV VERMONT, DEPT MICROBIOL & MOLEC GENET, GIVEN BLDG, BURLINGTON, VT 05405 USA
关键词
D O I
10.2307/3577926
中图分类号
Q [生物科学];
学科分类号
07 ; 0710 ; 09 ;
摘要
The inactivation efficiency and repair of single-strand breaks was investigated using model strand breaks created by endonucleolytic incision of damaged DNA. ∅X-174 duplex transfecting DNA containing either thymine glycols, urea residues, or abasic (AP) sites was incubated with AP endonucleases that produce breaks on the 3' side, the 5' side, or both sides of the lesion. For each lesion, incubation with Escherichia coli endonuclease III results in a single-strand break containing a 3' α,β-unsaturated aldehyde (4-hydroxy-2-pentenal), while treatment of AP- or urea-containing DNA with E. coli endonuclease IV results in a single-strand break containing a 5' deoxyribose or a 5' deoxyribosylurea moiety, respectively. Incubation of lesion-containing DNA with both enzymes results in a base gap. Ligatable nicks containing 3' hydroxyl and 5' phosphate moieties were produced by subjecting undamaged DNA to DNase I. When the biological activity of these DNAs was assessed in wild-type cells, ligatable nicks were not lethal, but each of the other strand breaks tested was lethal, having inactivation efficiencies between 0.12 and 0.14. These inactivation efficiencies are similar to those of the base lesions from which the strand breaks were derived. In keeping with the current model of base excision repair, when ∅X duplex DNA containing strand breaks with a blocked 3' terminus was transfected into an E. coli double mutant lacking the major 5' cellular AP endonucleases, a greater than twofold decrease in survival was observed. Moreover, when this DNA was treated with a 5' AP endonuclease prior to transfection, the survival returned to that of wild type. As expected, when DNA containing strand breaks with a 5' blocked terminus or DNA containing base gaps was transfected into the double mutant lacking 5' AP endonucleases, the survival was the same in wild-type cells. The decreased survival of transfecting DNA containing thymine glycols, urea, or AP sites observed in appropriate base excision repair-defective mutants was also obviated if the DNA was incubated with the homologous enzyme prior to transfection. Thus, in every case, with both base lesions and single-strand breaks, the lesion was repaired in the cell by the enzyme that recognizes it in vitro. Furthermore, the repair step in the cell could be eliminated if the appropriate enzyme was added in vitro prior to transfection. Also, the fact that strand breaks with blocked 3' or 5' termini as well as base gaps had inactivation efficiencies equal to each other and to the base damages from which they were derived suggests that the rate-limiting step in the base excision repair of damaged ∅X DNA is repair polymerization.
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页码:357 / 366
页数:10
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