John W. Chase

Intermediates in homologous pairing promoted by recA protein: Isolation and characterization of active presynaptic complexes

recA protein promotes homologous pairing and strand exchange by an ordered reaction in which the protein first polymerizes on single-stranded DNA. This presynaptic intermediate, which can be formed either in the presence or absence of Escherichia coli single-stranded binding protein (SSB), has been isolated by gel filtration and characterized. At saturation, purified complexes contained one molecule of recA protein per 3.6 nucleotide residues of single-stranded DNA. Complexes that had been formed in the presence of SSB contained up to one molecule of SSB per 15 nucleotide residues, but the content of SSB in different preparations of isolated complexes appeared to be inversely related to the content of recA protein. Even when they have lost as much as a third of their recA protein, presynaptic complexes can retain activity, because the formation of stable joint molecules depends principally on the binding of recA protein to the single-stranded DNA in the localized region that corresponds to the end of the duplex substrate.

Tryptophan 54 and phenylalanine 60 are involved synergistically in the binding of E. coli SSB protein to single-stranded polynucleotides

The binding of both wild‐type and point‐mutated E. coli single‐stranded DNA‐binding (SSB) protein to poly(deoxythymidylic acid) has been studied by fluorescence and optical detection of triplet state magnetic resonance spectroscopy. Involvement of tryptophan residues 40 and 54 in stacking interactions with nucleotide bases has been inferred earlier from such studies. Investigation of a point mutation in the E. coli SSB gene product obtained by site specific oligonucleotide mutagenesis in which Phe‐60 is replaced by alanine strongly suggests the participation of Phe‐60 in the binding process, possibly by the formation of an extended stacking structure by Trp‐54, thymine and Phe‐60. This hypothesis is supported by results on the point mutations in which His‐55 is replaced by either leucine or tyrosine.

Crystals of Escherichia coli single-strand DNA-binding protein show that the tetramer has D2 symmetry.

Crystals of Escherichia coli single-strand DNA-binding protein (SSB) that are suitable for high-resolution. X-ray crystallographic studies have been obtained. Form I crystals are monoclinic space group C 2 with unit cell dimensions of a = 105·5 A, b = 62·7 A, c = 98·1 A, β = 112·6°, and with an asymmetric unit containing two native and two proteolytically degraded SSB subunits. Form II crystals are hexagonal space group P 6 2(4) 22 with a = b = 61·4 A and c = 264·3 A and with two chymotryptic fragment SSB subunits per asymmetric unit. Calculation of rotation function using 6 A data shows that SSB has D 2 symmetry.

DNA repair in E. coli strains deficient in single-strand DNA binding protein

SummaryWeigle reactivation and mutagenesis have been found to be defective in strains of E. coli deficient in single-strand DNA binding protein (SSB). These defects parallel those previously found in prophage induction and amplification of recA protein synthesis in ssb strains. Together, these results demonstrate a role for SSB in the induction of SOS responses. UV survival studies of ssb- recA- and ssb- uvr- strains are presented which also suggest a role for SSB in recombinational repair processes but not in excision repair. Studies of host cell reactivation support this latter conclusion.

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 lambda +, 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.

DNA repair properties of Escherichia coli tif-1, recAo281 and lexA1 strains deficient in single-strand DNA binding protein

SummaryMutations affecting single-strand DNA binding protein (SSB) impair induction of mutagenic (SOS) repair. To further investigate the role of SSB in SOS induction and DNA repair, isogenic strains were constructed combining the ssb+, ssb-1 or ssb-113 alleles with one or more mutations known to alter regulation of damage inducible functions. As is true in ssb+ strains tif-1 (recA441) was found to allow thermal induction of prophage λ+ and Weigle reactivation in ssb-1 and ssb-113 strains. Furthermore, tif-1 decreased the UV sensitivity of the ssb-113 strain slightly and permitted UV induction of prophage λ+ at 30°C. Strains carrying the recAo281 allele were also constructed. This mutation causes high constitutive levels of RecA protein synthesis and relieves much of the UV sensitivity conferred by lexA− alleles without restoring SOS (error-prone) repair. In contrast, the recAo281 allele failed to alleviate the UV sensitivity associated with either ssb− mutation. In a lexA1 recAo281 background the ssb-1 mutation increased the extent of postirradiation DNA degradation and concommitantly increased UV sensitivity 20-fold to the level exhibited by a recA1 strain. The ssb-113 mutation also increased UV sensitivity markedly in this background but did so without greatly increasing postirradiation DNA degradation. These results suggest a direct role for SSB in recombinational repair apart from and in addition to its role in facilitating induction of the recA-lexA regulon.

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

Abstract 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.

Repair resynthesis in Escherichia coli mutants deficients 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.

PYRIMIDINE DIMER EXCISION IN EXONUCLEASE DEFICIENT MUTANTS OF ESCHERICHIA COLI

ABSTRACT The rate of pyrimidine dimer excision has been measured in Escherichia coli strains deficient in exonuclease V, exonuclease VII, and the 5′→3′ exonuclease of DNA polymerase I. The results suggest that a reduced level of the 5′→3′ exonuclease of DNA polymerase I diminishes the rate of dimer excision and that an additional deficiency in exonuclease VII causes a significantly greater reduction in the cells ability to remove dimers.