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author:("pariza, R R")
1.  Reversible protein phosphorylation modulates nucleotide excision repair of damaged DNA by human cell extracts. 
Nucleic Acids Research  1996;24(3):433-440.
Nucleotide excision repair of DNA in mammalian cells uses more than 20 polypeptides to remove DNA lesions caused by UV light and other mutagens. To investigate whether reversible protein phosphorylation can significantly modulate this repair mechanism we studied the effect of specific inhibitors of Ser/Thr protein phosphatases. The ability of HeLa cell extracts to carry out nucleotide excision repair in vitro was highly sensitive to three toxins (okadaic acid, microcystin-LR and tautomycin), which block PP1- and PP2A-type phosphatases. Repair was more sensitive to okadaic acid than to tautomycin, suggesting the involvement of a PP2A-type enzyme, and was insensitive to inhibitor-2, which exclusively inhibits PP1-type enzymes. In a repair synthesis assay the toxins gave 70% inhibition of activity. Full activity could be restored to toxin-inhibited extracts by addition of purified PP2A, but not PP1. The p34 subunit of replication protein A was hyperphosphorylated in cell extracts in the presence of phosphatase inhibitors, but we found no evidence that this affected repair. In a coupled incision/synthesis repair assay okadaic acid decreased the production of incision intermediates in the repair reaction. The formation of 25-30mer oligonucleotides by dual incision during repair was also inhibited by okadaic acid and inhibition could be reversed with PP2A. Thus Ser/Thr- specific protein phosphorylation plays an important role in the modulation of nucleotide excision repair in vitro.
PMCID: PMC145647  PMID: 8602355
2.  Activation of multiple antibiotic resistance and binding of stress-inducible promoters by Escherichia coli Rob protein. 
Journal of Bacteriology  1995;177(7):1655-1661.
Multiple antibiotic resistance in Escherichia coli can be mediated by induction of the SoxS or MarA protein, triggered by oxygen radicals (in the soxRS regulon) or certain antibiotics (in the marRAB regulon), respectively. These small proteins (SoxS, 107 residues; MarA, 127 residues) are homologous to the C terminus of the XylS-AraC family of proteins and are more closely related to a approximately 100-residue segment in the N terminus of Rob protein, which binds the right arm of the replication origin, oriC. We investigated whether the SoxS-MarA homology in Rob might extend to the regulation of some of the same inducible genes. Overexpression of Rob indeed conferred multiple antibiotic resistance similar to that known for SoxS and MarA (against chloramphenicol, tetracycline, nalidixic acid, and puromycin), as well as resistance to the superoxide-generating compound phenazine methosulfate. The Rob-induced antibiotic resistance depended only partially on the micF antisense RNA that down-regulates the OmpF outer membrane porin to limit antibiotic uptake. Similar antibiotic resistance was conferred by expression of a Rob fragment containing only the N-terminal 123 residues that constitute the SoxS-MarA homology. Both intact Rob and the N-terminal fragment activated expression of stress genes (inaA, fumC, sodA) but with a pattern distinct from that found for SoxS and MarA. Purified Rob protein bound a DNA fragment containing the micF promoter (50% bound at approximately 10(-9) M Rob) as strongly as it did oriC, and it bound more weakly to DNA containing the sodA, nfo, or zwf promoter (50% bound at 10(-8) to 10(-7) M). Rob formed multiple DNA-protein complexes with these fragments, as seen previously for SoxS. These data point to a DNA-binding gene activator module used in different protein contexts.
PMCID: PMC176790  PMID: 7896685
3.  Repressor mutations in the marRAB operon that activate oxidative stress genes and multiple antibiotic resistance in Escherichia coli. 
Journal of Bacteriology  1994;176(1):143-148.
Resistance to multiple antibiotics and certain oxidative stress compounds was conferred by three independently selected mutations (marR1, soxQ1, and cfxB1) that mapped to 34 min on the Escherichia coli chromosome. Mutations at this locus can activate the marRAB operon, in which marR encodes a putative repressor of mar transcription and marA encodes a putative transcriptional activator of defense genes against antibiotics and oxidants. Overexpression of the wild-type MarR protein reversed the phenotypes (antibiotic resistance and increased antioxidant enzyme synthesis) of all three mutants. DNA sequence analysis showed that, like marR1, the other two mutations were alterations of marR: a 285-bp deletion in cfxB1 and a GC-->AT transition at codon 70 (Ala-->Thr) in soxQ1. All three mutations cause increased amounts of mar-specific RNA, which supports the hypothesis that MarR has a repressor function in the expression of the marRAB operon. The level of mar RNA was further induced by tetracycline in both the marR1 and soxQ1 strains but not in the cfxB1 deletion mutant. In the cfxB1 strain, the level of expression of a truncated RNA, with or without tetracycline exposure, was the same as the fully induced level in the other two mutants. Overproduction of MarR in the cfxB1 strain repressed the transcription of the truncated RNA and restored transcriptional inducibility by tetracycline. Thus, induction of the marRAB operon results from the relief of the repression exerted by MarR. The marRAB operon evidently activates both antibiotic resistance and oxidative stress genes.
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PMCID: PMC205025  PMID: 8282690

Results 1-3 (3)