RESUMO
We investigate the dynamical properties of the non-heme iron (NHFe) in His-tagged photosynthetic bacterial reaction centers (RCs) isolated from Rhodobacter (Rb.) sphaeroides. Mössbauer spectroscopy and nuclear inelastic scattering of synchrotron radiation (NIS) were applied to monitor the arrangement and flexibility of the NHFe binding site. In His-tagged RCs, NHFe was stabilized only in a high spin ferrous state. Its hyperfine parameters (IS=1.06±0.01mm/s and QS=2.12±0.01mm/s), and Debye temperature (θ(D0)~167K) are comparable to those detected for the high spin state of NHFe in non-His-tagged RCs. For the first time, pure vibrational modes characteristic of NHFe in a high spin ferrous state are revealed. The vibrational density of states (DOS) shows some maxima between 22 and 33meV, 33 and 42meV, and 53 and 60meV and a very sharp one at 44.5meV. In addition, we observe a large contribution of vibrational modes at low energies. This iron atom is directly connected to the protein matrix via all its ligands, and it is therefore extremely sensitive to the collective motions of the RC protein core. A comparison of the DOS spectra of His-tagged and non-His-tagged RCs from Rb. sphaeroides shows that in the latter case the spectrum was overlapped by the vibrations of the heme iron of residual cytochrome c(2), and a low spin state of NHFe in addition to its high spin one. This enabled us to pin-point vibrations characteristic for the low spin state of NHFe.
Assuntos
Ferro/química , Complexo de Proteínas do Centro de Reação Fotossintética/química , Rhodobacter sphaeroides/química , Sítios de Ligação , Transporte de Elétrons , Ferro/metabolismo , Cinética , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Rhodobacter sphaeroides/crescimento & desenvolvimento , Rhodobacter sphaeroides/metabolismo , Espectroscopia de Mossbauer , Síncrotrons , VibraçãoRESUMO
We have studied the ability of yeast DNA polymerases to carry out repair of lesions caused by UV irradiation in Saccharomyces cerevisiae. By the analysis of postirradiation relative molecular mass changes in cellular DNA of different DNA polymerases mutant strains, it was established that mutations in DNA polymerases delta and epsilon showed accumulation of single-strand breaks indicating defective repair. Mutations in other DNA polymerase genes exhibited no defects in DNA repair. Thus, the data obtained suggest that DNA polymerases delta and epsilon are both necessary for DNA replication and for repair of lesions caused by UV irradiation. The results are discussed in the light of current concepts concerning the specificity of DNA polymerases in DNA repair.
Assuntos
DNA Polimerase III/metabolismo , DNA Polimerase II/metabolismo , Reparo do DNA , DNA Fúngico/efeitos da radiação , Saccharomyces cerevisiae/efeitos da radiação , Dano ao DNA , Reparo do DNA/genética , Mutação , Saccharomyces cerevisiae/genética , Raios UltravioletaRESUMO
In the yeast Saccharomyces cerevisiae three different DNA polymerases alpha, delta and epsilon are involved in DNA replication. DNA polymerase alpha is responsible for initiation of DNA synthesis and polymerases delta and epsilon are required for elongation of DNA strand during replication. DNA polymerases delta and epsilon are also involved in DNA repair. In this work we studied the role of these three DNA polymerases in the process of recombinational synthesis. Using thermo-sensitive heteroallelic mutants in genes encoding DNA polymerases we studied their role in the process of induced gene conversion. Mutant strains were treated with mutagens, incubated under permissive or restrictive conditions and the numbers of convertants obtained were compared. A very high difference in the number of convertants between restrictive and permissive conditions was observed for polymerases alpha and delta, which suggests that these two polymerases play an important role in DNA synthesis during mitotic gene conversion. Marginal dependence of gene conversion on the activity of polymerase epsilon indicates that this DNA polymerase may be involved in this process but rather as an auxiliary enzyme.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Conversão Gênica , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Dano ao DNA , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Reparo do DNA , DNA Fúngico/biossíntese , DNA Fúngico/genética , DNA Polimerase Dirigida por DNA/genética , Genes Fúngicos/efeitos dos fármacos , Genes Fúngicos/efeitos da radiação , Metanossulfonato de Metila , Mutagênicos , Mutação , Recombinação Genética , Trioxsaleno , Raios UltravioletaRESUMO
The ability of four yeast DNA polymerase mutant strains to carry out the repair of DNA treated with MMS was studied. Mutation in DNA polymerase Rev3, as well as the already known mutation in the catalytic subunit of DNA polymerase delta, were both found to lead to the accumulation of single-strand breaks, which indicates defective repair. A double-mutant strain carrying mutations in DNA polymerase delta and a deletion in the REV3 gene had a complete repair defect, both at permissive (23 degrees C) and restrictive (38 degrees C) temperatures, which was not observed in other pairwise combinations of tested polymerase mutants. Other polymerases are not involved in the repair of exogenous DNA methylation damage, since neither mutation in the DNA polymerase epsilon, nor deletion in the DNA polymerase IV (beta70) gene, caused defective repair. The data obtained suggest that DNA polymerases delta and Rev3p are both necessary to perform repair synthesis in the base-excision repair of methylation damage. The results are discussed in the light of current concepts on the role of DNA polymerase Rev3 in mutagenesis.