Biomedical subjects
I Tessman
Publications and source records attributed to I Tessman.
Secretory otitis media: the Cantekin affair.
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Mechanism of SOS mutagenesis of UV-irradiated DNA: mostly error-free processing of deaminated cytosine.
We measured the kinetics of growth and mutagenesis of UV-irradiated DNA of phages S13 and lambda that were undergoing SOS repair; the kinetics strongly suggest that most of SOS mutagenesis arises from the deamination of cytosine in cyclobutane pyrimidine dimers, producing C----T transitions. This occurs because the SOS mechanism bypasses T--T dimers promptly, while bypass of cytosine-containing dimers is delayed long enough for deamination to occur. The mutations are thus primarily the product of a faithful mechanism of lesion bypass by a DNA polymerase and are not, as had been generally thought, the product of an error-prone mechanism. All of these observations are explained by the A-rule, which is that adenine nucleotides are inserted noninstructionally opposite DNA lesions.
Documentation of the claim that modulation of UV light pulses increases the bactericidal effectiveness of the light.
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Further evidence that transposition of Tn5 in Escherichia coli is strongly enhanced by constitutively activated RecA proteins.
We have shown that excision and transposition of Tn5 in Escherichia coli are greatly increased by recA(Prtc) genes, which encode constitutively activated RecA proteins (C.-T. Kuan, S.-K. Liu, and I. Tessman, Genetics 128:45-57, 1991). Contrary results, showing a significant decrease in Tn5 transposition under SOS conditions, were subsequently reported (M. D. Weinreich, J. C. Makris, and W. S. Reznikoff, J. Bacteriol. 173:6910-6918, 1991). We have extended our studies to examine the following: (i) transposition of Tn5 from sites in the phoA, phoB, proC, trpD, and ilvD genes; (ii) the effect of gene transcription; (iii) the comparative effect of dinD+ and dinD(Def) alleles; (iv) the use of a mating-out assay of transposition; (v) the effect of a recA(Prtc) allele located at the normal chromosomal site; and (vi) the effect at 41.5 degrees C of the recA441(Prtc) allele. The new results fully confirm our previous conclusions, including the fact that the frequency of Tn5 transposition under constitutive SOS conditions is site dependent.
The risk of HIV-1 in screened blood donations.
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The two-step model of UV mutagenesis reassessed: deamination of cytosine in cyclobutane dimers as the likely source of the mutations associated with photoreactivation.
A large increase in the incidence of bacteriophage mutants is found after photoreactivation of UV-irradiated phage S13. The increase was seen only when the irradiated phage were stored before they were photoreactivated; the maximum mutation frequency was achieved after storage for 2 h at 4 degrees C or 30 min at 37 degrees C. The mutations can be attributed entirely to deamination of cytosine in cyclobutane dimers. Naked S13 DNA was stored for 2 h at 37 degrees C after being irradiated with wavelengths greater than or equal to 290 nm in the presence of 0.2% acetophenone, which sensitizes the formation of thymine-thymine but not cytosine-containing dimers; the specific mutation frequency was 7.2-fold lower compared to the frequency produced by irradiation in the absence of the photosensitizer, confirming that cytosine dimers are a major source of mutations. These results undermine the basis for the two-step model of UV mutagenesis in which a distinctly separate misincorporation step is supposed to precede the lesion bypass step; instead the results support a different two-step model, in which a deamination step precedes the bypass. The S13 capsid appears to completely inhibit the putative deamination reaction at about 75% of the dimer sites.
Excision and transposition of Tn5 as an SOS activity in Escherichia coli.
Excision and transposition of the Tn5 element in Escherichia coli ordinarily appear to occur by recA-independent mechanisms. However, recA(Prtc) genes, which encode RecA proteins that are constitutively activated to the protease state, greatly enhanced excision and transposition; both events appeared to occur concomitantly and without destruction of the donor DNA. The recombinase function of the RecA protein was not required. Transposition was accompanied by partial, and occasionally full, restoration of the functional integrity of the gene vacated by the excised Tn5. The stimulation of transposition was inhibited by an uncleavable LexA protein and was strongly enhanced by an additional role of the RecA(Prtc) protein besides its mediation of LexA cleavage. To account for the enhanced transposition, we suggest that (i) there may be a LexA binding site within the promoter for the IS50 transposase, (ii) activated RecA may cleave the IS50 transposition inhibitor, and (iii) the transposase may be formed by RecA cleavage of a precursor molecule.
LexA protein of Escherichia coli represses expression of the Tn5 transposase gene.
The LexA protein of Escherichia coli represses expression of a variety of genes that, by definition, constitute the SOS regulon. Genetic evidence suggests that Tn5 transposition is also regulated by the product of the lexA gene (C.-T. Kuan, S.-K. Liu, and I. Tessman, Genetics 128:45-57, 1991). We now show that the LexA protein represses expression of the tnp gene, located in the IS50R component of Tn5, which encodes a transposase, and that LexA does not repress expression of the IS50R inh gene, which encodes an inhibitor of transposition. Elimination of LexA resulted in increased expression of the tnp gene by a factor of 2.7 +/- 0.4, as indicated by the activity of a lacZ gene fused to the tnp gene. LexA protein retarded the electrophoretic movement of a 101-bp segment of IS50R DNA that contained a putative LexA protein-binding site in the tnp promoter; the interaction between the LexA repressor and the promoter region of the tnp gene appears to be relatively weak. These features show that the IS50R tnp gene is a member of the SOS regulon.
Error-prone SOS repair can be error-free.
Most of the mutagenesis that accompanies the SOS repair of ultraviolet light-induced lesions in the single-stranded DNA of phage S13 is eliminated when the groES or the groEL gene of Escherichia coli is defective. Therefore, this SOS mutagenesis is not a necessary consequence of what is commonly called error-prone repair, but is additionally imposed on the repair system by the GroE heat shock proteins, which are responsible for the assembly of polypeptides into multimeric structures.
Mutagenesis by proximity to the recA gene of Escherichia coli.
Escherichia coli recA (Prtc) strains, which produce protease constitutive RecA proteins in the absence of DNA-damaging treatments, display an increased frequency of spontaneous mutations. These mutations occurred preferentially in the neighborhood of the recA gene. This cis-like mutagenic effect was observed in the recA, rexAB, phoE and bio genes. The localized mutagenesis can be explained by the ease with which RecA(Prtc) proteins are activated to the protease state, which implies that there should be a relatively high concentration of activated RecA protein near the recA gene, where the protein is synthesized. The unusually high frequency of mutation in the recA gene is a novel example of an overactive gene preferentially turning itself down by mutation.
groE genes affect SOS repair in Escherichia coli.
Repair of UV-irradiated bacteriophage in Escherichia coli by Weigle reactivation requires functional recA+ and umuD+C+ genes. When the cells were UV irradiated, the groE heat shock gene products, GroES and GroEL, were needed for at least 50% of the Weigle reactivation of the single-stranded DNA phage S13. Because of repression of the umuDC and recA genes, Weigle reactivation is normally blocked by the lexA3(Ind-) mutation (which creates a noncleavable LexA protein), but it was restored by a combination of a high-copy-number umuD+C+ plasmid and a UV dose that increases groE expression. Maximal reactivation was achieved by elevated amounts of the Umu proteins, which was accomplished in part by UV-induced expression of the groE genes. By increasing the number of copies of the umuD+C+ genes, up to 50% of the normal amount of reactivation of S13 was achieved in an unirradiated recA+ host.
SOS repair can be about as effective for single-stranded DNA as for double-stranded DNA and even more so.
As ordinarily measured, the SOS repair of damaged DNA by Weigle reactivation appears to be more effective for double-stranded (ds) than for single-stranded (ss) DNA bacteriophages. A complicating feature, which is usually not considered, is the possibility of DNA-protein cross-linking of ssDNA to the viral capsid, which would conceivably be an extraneous source of nonreactivable lesions. This idea is supported in studies of phage S13 by the observation that photoreactivation more than doubles when naked ssDNA is substituted for encapsidated ssDNA as the UV target. The same effect was observed for Weigle reactivation; there was little, if any, difference in the reactivation of ssDNA and dsDNA when naked DNA was irradiated. Moreover, in a uvrA mutant, ssDNA actually had the advantage; Weigle reactivation was then more than twice as effective for ssDNA as for dsDNA. It is also shown that when a suitable measure of Weigle mutagenesis is used, there is no convincing evidence that dsDNA is mutagenized more effectively than ssDNA.
Limited significance of null results.
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Levels of DNA topoisomerases, single-stranded-DNA-binding protein, and DNA polymerase I in rho+ and rho-15 strains of Escherichia coli.
The Escherichia coli rho-15 mutant, which is highly defective in transcription termination, was examined to see whether its reduced DNA superhelicity could be explained by altered expression of proteins that may affect DNA structure. Levels of DNA gyrase and topoisomerase I were normal; levels of single-stranded-DNA-binding protein, DNA polymerase I, and a protein tentatively identified as Lon were significantly altered.
Activation of protease-constitutive recA proteins of Escherichia coli by all of the common nucleoside triphosphates.
To understand why the RecA proteins of the protease-constitutive recA1202 and recA1211 mutants show very high protease activities in vivo without the usual need for DNA damage (E. S. Tessman and P. Peterson, J. Bacteriol. 163:677-687, 1985), we examined the activation of the mutant proteins by nucleoside triphosphates (NTPs) in vitro. In vivo, the mutant protease activities are resistant to inhibition by cytidine plus guanosine (C + G) in the growth medium, in contrast to the activities of weaker mutants, such as recA441, which are sensitive to C + G inhibition. We found that RecA1202 and RecA1211 proteins, in contrast to RecA+, can use natural NTPs other than ATP and dATP as cofactors in the cleavage of LexA repressor. The effectiveness of NTPs in promoting LexA cleavage by RecA1202 and RecA1211 proteins decreased in roughly the following order: dATP greater than ATP greater than UTP greater than ATP-gamma S greater than dCTP greater than CTP greater than dGTP greater than GTP greater than TTP. These mutant proteins showed higher affinities for ATP and single-stranded DNA and higher repressor cleavage activities than RecA+ protein. With the various effectors (single-stranded DNA or NTPs), the RecA1202 protein always showed more activity than RecA1211 in the cleavage of LexA repressor in vitro, which is consistent with the greater activity of the recA1202 mutant in vivo. The results explain, in part, why some recA mutants have unusually high constitutive RecA protease activity and why that activity is more or less resistant to C + G inhibition.
Activation of protease-constitutive recA proteins of Escherichia coli by rRNA and tRNA.
The RecA proteins of the unusually strong protease-constitutive mutants recA1202 and recA1211 can use RNA in addition to single-stranded DNA (ssDNA) as a cofactor in the cleavage of the LexA repressor in vitro. In the presence of rRNA or tRNA, the effectiveness of these proteins decreased in the order RecA1202 greater than RecA1211 much greater than RecA+, which is also the order of their in vivo constitutive protease activities. The effectiveness of rRNA was comparable to that of ssDNA in the cleavage of the LexA repressor by either mutant protease. Although all the common nucleoside triphosphates can act as positive effectors for LexA cleavage by the two mutant proteins in the presence of ssDNA (W. B. Wang, M. Sassanfar, I. Tessman, J. W. Roberts, and E. S. Tessman, J. Bacteriol. 170:4816-4822, 1988), only dATP, ATP, and ATP-gamma-S were effective in the presence of RNA. Our results explain more fully why certain recA mutants have high constitutive protease activities in vivo.