Repair resynthesis in Escherichia coli mutants deficient in single-stranded DNA-binding protein

A series of Escherichia coli strains deficient in single-stranded DNA-binding protein (SSB) and DNA polymerase I was constructed in order to analyze the effects of these mutations on DNA repair resynthesis after UV-irradiation. Since SSB has been suggested to play a role in protecting single-stranded regions which may transiently exist during excision repair and since long single-stranded regions are believed to occur frequently as repair intermediates in strains deficient in DNA polymerase I, studies of repair resynthesis and strand rejoining were performed on strains containing both the ssb-1 and polA1 mutations. Repair resynthesis appears to be slightly decreased in the ssb-1 strain at 42 degrees C relative to the wild-type; however, this effect is not enhanced in a polA1 derivative of this strain. After UV-irradiation, the single-strand molecular weight of the DNA of an ssb-1 strain decreases and fails to recover to normal size. These results are discussed in the context of long patch repair as an inducible component of repair resynthesis and of the protection of intermediates in the excision repair process by SSB. A direct role for SSB in repair resynthesis involving modulation of the proteins involved in this mode of DNA synthesis (particularly stimulation of DNA polymerase II) is not supported by our findings.     

A filter assay specific to Eco helix-destabilizing Protein I in crude extracts

A filter assay for Eco helix-destabilizing Protein I is reported and shown by immunological analysis specifically to detect Eco HD Protein I in crude extracts. The assay detects no activity in strains carrying a temperature-sensitive allele for Eco HD Protein I. The assay is linear over a 20-fold range and can reliably detect a nanogram or less of Eco HD Protein I.     

Amplification of ssb-1 mutant single-stranded DNA-binding protein in Escherichia coli

The ssb-1 gene encoding a mutant Escherichia coli single-stranded DNA-binding protein has been cloned into plasmid pACYC184. The amount of overproduction of the cloned ssb-1 gene is dependent upon its orientation in the plasmid. In the less efficient orientation, 25-fold more mutant protein is produced than in strains carrying only one (chromosomal) copy of the gene: the other orientation results in more than 60-fold overproduction of this protein. Analysis of the effects of overproduction of the ssb-1 encoded protein has shown that most of the deficiencies associated with the ssb-1 mutation when present in single gene copy, including temperature-sensitive conditional lethality and deficiencies in amplified synthesis of RecA protein and ultraviolet light-promoted induction of prophage λ⁺, are reversed by increased production of ssb-1 mutant protein. These results provide evidence in vivo that SSB protein plays an active role in recA-dependent processes. Homogenotization of a nearby genetic locus (uvrA) was identified in the cloning of the ssb-1 mutant gene. This observation has implications in the analysis of uvrA^? mutant strains and will provide a means of transferring ssb^? mutations from plasmids to the chromosome. On a broader scale, the observation may provide the basis of a general strategy to transfer mutations between plasmids and chromosomes.