The oxidation of methionine-54 of epoetinum alfa does not affect molecular structure or stability, but does decrease biological activity.

Erythropoietin therapy is used to treat severe anemia in renal failure and chemotherapy patients. One of these therapies based on recombinant human erythropoietin is marketed under the trade name of EPREX and utilizes epoetinum alfa as the active pharmaceutical ingredient. The effect of oxidation of methionine-54 on the structure and stability of the erythropoietin molecule has not been directly tested. We have observed partial and full chemical oxidation of methionine-54 to methionine-54 sulfoxide, accomplished using tert-Butylhydroperoxide and hydrogen peroxide, respectively. A blue shift in the fluorescence center of spectral mass wavelength was observed as a linear response to the level of methionine sulfoxide in the epoetinum alfa molecule, presumably arising from a local change in the environment near tryptophan-51, as supported by potassium iodide quenching studies. Circular dichroism studies demonstrated no change in the folded structure of the molecule with methionine oxidation. The thermal unfolding profiles of partial and completely oxidized epoetinum alfa overlap, with a T(m) of 49.5 degrees C across all levels of methionine sulfoxide content. When the protein was tested for activity, a decrease in biological activity was observed, correlating with methionine sulfoxide levels. An allosteric effect between Met54, Trp51, and residues involved in receptor binding is proposed. These results indicate that methionine oxidation has no effect on the folded structure and global thermodynamic stability of the recombinant human erythropoietin molecule. Oxidation can affect potency, but only at levels significantly in excess of those seen in EPREX.

Separation of mutation avoidance and antirecombination functions in an Escherichia coli mutS mutant.

DNA mismatch repair in Escherichia coli has been shown to be involved in two distinct processes: mutation avoidance, which removes potential mutations arising as replication errors, and antirecombination which prevents recombination between related, but not identical (homeologous), DNA sequences. We show that cells with the mutSDelta800 mutation (which removes the C-terminal 53 amino acids of MutS) on a multicopy plasmid are proficient for mutation avoidance. In interspecies genetic crosses, however, recipients with the mutSDelta800 mutation show increased recombination by up to 280-fold relative to mutS+. The MutSDelta800 protein binds to O6-methylguanine mismatches but not to intrastrand platinated GG cross-links, explaining why dam bacteria with the mutSDelta800 mutation are resistant to cisplatin, but not MNNG, toxicity. The results indicate that the C-terminal end of MutS is necessary for antirecombination and cisplatin sensitization, but less significant for mutation avoidance. The inability of MutSDelta800 to form tetramers may indicate that these are the active form of MutS.

MutS inhibits RecA-mediated strand transfer with methylated DNA substrates.

DNA mismatch repair (MMR) sensitizes human and Escherichia coli dam cells to the cytotoxic action of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) while abrogation of such repair results in drug resistance. In DNA methylated by MNNG, MMR action is the result of MutS recognition of O6-methylguanine base pairs. MutS and Ada methyltransferase compete for the MNNG-induced O6-methylguanine residues, and MMR-induced cytotoxicity is abrogated when Ada is present at higher concentrations than normal. To test the hypothesis that MMR sensitization is due to decreased recombinational repair, we used a RecA-mediated strand exchange assay between homologous phiX174 substrate molecules, one of which was methylated with MNNG. MutS inhibited strand transfer on such substrates in a concentration-dependent manner and its inhibitory effect was enhanced by MutL. There was no effect of these proteins on RecA activity with unmethylated substrates. We quantified the number of O6-methylguanine residues in methylated DNA by HPLC-MS/MS and 5-10 of these residues in phiX174 DNA (5386 bp) were sufficient to block the RecA reaction in the presence of MutS and MutL. These results are consistent with a model in which methylated DNA is perceived by the cell as homeologous and prevented from recombining with homologous DNA by the MMR system.

Spontaneous and cisplatin-induced recombination in Escherichia coli.

To measure cisplatin (cis-diaminodichloroplatinum(II))-induced recombination, we have used a qualitative intrachromosomal assay utilizing duplicate inactive lac operons containing non-overlapping deletions and selection for Lac+ recombinants. The two operons are separated by one Mb and conversion of one of them yields the Lac+ phenotype. Lac+ formation for both spontaneous and cisplatin-induced recombination requires the products of the recA, recBC, ruvA, ruvB, ruvC, priA and polA genes. Inactivation of the recF, recO, recR and recJ genes decreased cisplatin-induced, but not spontaneous, recombination. The dependence on PriA and RecBC suggests that recombination is induced following stalling or collapse of replication forks at DNA lesions to form double strand breaks. The lack of recombination induction by trans-DDP suggests that the recombinogenic lesions for cisplatin are purine-purine intrastrand crosslinks.

Differential effects of cisplatin and MNNG on dna mutants of Escherichia coli.

DNA mismatch repair (MMR) in mammalian cells or Escherichia coli dam mutants increases the cytotoxic effects of cisplatin and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We found that, unlike wildtype, the dnaE486 (alpha catalytic subunit of DNA polymerase III holoenzyme) mutant, and a DnaX (clamp loader subunits) over-producer, are sensitive to cisplatin but resistant to MNNG at the permissive temperature for growth. Survival of dam-13 dnaN159 (beta sliding clamp) bacteria to cisplatin was significantly less than dam cells, suggesting decreased MMR, which may be due to reduced MutS-beta clamp interaction. We also found an elevated spontaneous mutant frequency to rifampicin resistance in dnaE486 (10-fold), dnaN159 (35-fold) and dnaX36 (10-fold) strains. The mutation spectrum in the dnaN159 strain was consistent with increased SOS induction and not indicative of MMR deficiency.

RecA dimers serve as a functional unit for assembly of active nucleoprotein filaments.

All RecA-like recombinase enzymes catalyze DNA strand exchange as elongated filaments on DNA. Despite numerous biochemical and structural studies of RecA and the related Rad51 and RadA proteins, the unit oligomer(s) responsible for nucleoprotein filament assembly and coordinated filament activity remains undefined. We have created a RecA fused dimer protein and show that it maintains in vivo DNA repair and LexA co-protease activities, as well as in vitro ATPase and DNA strand exchange activities. Our results support the idea that dimeric RecA is an important functional unit both for assembly of nucleoprotein filaments and for their coordinated activity during the catalysis of homologous recombination.

The MutS C terminus is essential for mismatch repair activity in vivo.

An Escherichia coli K-12 strain was constructed with a chromosomal deletion (mutSdelta800) in the mutS gene that produced the removal of the C-terminal 53 amino acids which are not present in the MutS crystal structure. This strain has a MutS null phenotype for mutation avoidance, anti-recombination, and sensitivity to cytotoxic agents in a dam mutant background.

MutS inhibits RecA-mediated strand exchange with platinated DNA substrates.

Human cell lines and Escherichia coli dam mutants are sensitive to the cytotoxic action of the anticancer agent, cisplatin. Introduction of mutations disabling DNA mismatch repair into these cell lines renders them resistant to the action of this drug. We used RecA-mediated strand exchange between homologous phiX174 molecules, one that was platinated and the other that was unmodified, to show that strand transfer is decreased in a dose-dependent manner. Transfer was severely decreased at 10 adducts per molecule (5,386 bp) and abolished with 24 adducts. At low levels of adduction, addition of MutS to the reaction further decreases the rate and yield in a dose-dependent manner. MutL addition was without effect even in the presence of MutS. The results suggest that although mismatch repair is beneficial for mutation avoidance, its antirecombination activity on inappropriate substrates can be lethal to the cell.

Regulated expression of the Escherichia coli dam gene.

Regulated expression of the Escherichia coli dam gene has been achieved with the araBAD promoter lacking a ribosome binding site. Cultures of dam mutants containing plasmid pMQ430 show no detectable methylation in the absence of arabinose and complete methylation in its presence. Dam methyltransferase is a substrate for the Lon protease.