2 research outputs found
The particulate methane monooxygenase from Methylococcus capsulatus (Bath)
No full textThe isolation procedure for the pMMO complex has been optimised to obtain a high
specific activity enzyme from Methylococcus capsulatus (Bath). The enzyme is
comprised of the pMMO hydroxylase (pMMOH) consisting of polypeptides 47,26 and
23kDa molecular mass. In addition to this, a putative pMMO reductase (pMMOR) was
also found to be necessary to maintain propylene oxidising activity. This component
was found to consist of two polypeptides of approximately 63 and 8kDa. Preliminary Nterminal
sequence data of the large subunit ofpMMOR indicates that the sequence bears
70% similarity to the methanol dehydrogenase (MDH) from Methylococcus capsulatus
(Bath). Therefore, we tentatively propose that the" MDH can act as a reductase
component to the pMMOH.
The significance of this result prompted investigations into the previous published
proposals that electrons derived from the methanol oxidation reaction can be channelled
back into the methane oxidation reaction by the methanol dehydrogenase, independent
of NADH. Any effect of methanol to act as a reductant to pMMO in membrane
preparations was lost upon isolation of the pMMO complex, indicating the necessity to
maintain a fully functional methanol dehydrogenase (MDH) upon isolation. In addition
to this, the in vitro electron donors of pMMO, NADH and duroquinol were found to act
via distinct pathways of electron transfer (electron transport inhibitor studies).
Electron paramagnetic resonance (EPR) spectroscopy data provided evidence that the
copper in the active site of pMMO existed as a mononuclear copper (II) centre not a
trinuclear copper centre suggested by Chan and coworkers (Chan et al., 1993; Nguyen et
al., 1994, 1996a, 1996b, 1998). In addition to this preliminary data also indicates the
presence of an iron centre which is only EPR visible after reduction of the complex
suggesting the majority of iron in the complex is EPR invisible. The exact nature of this
iron centre is still unclear.
A structural study of the pMMO complex has also been undertaken using electron
microscopy studies in conjunction with single particle analysis. This allowed low
resolution projection maps of different views of the pMMO complex to be generated.
The complex appears to exist in a polymeric state of at least a dimer, possibly a tetramer
if the molecular weight analysis calculated by sedimentation equilibrium analysis is
taken into account.
This study has provided some insight into basic characteristics and the structure of a
duroquinol-driven pMMO complex and its interaction with other electron transfer
proteins
Potential Role for 53BP1 in DNA End-joining Repair through Direct Interaction with DNA
No full textUpon DNA damage, p53-binding protein 1 (53BP1) relocalizes to sites of DNA double-strand breaks and forms discrete nuclear foci, suggesting its role in DNA damage responses. We show that 53BP1 changed its localization from the detergent soluble to insoluble fraction after treatment of cells with x-ray, but not with ultraviolet or hydroxyurea. Either DNase or phosphatase treatment of the insoluble fraction released 53BP1 into the soluble fraction, showing that 53BP1 binds to chromatin in a phosphorylation-dependent manner after X-irradiation of cells. 53BP1 was retained at discrete nuclear foci in X-irradiated cells even after detergent extraction of cells, showing that the chromatin binding of 53BP1 occurs at sites of DNA double-strand breaks. The minimal domain for focus formation was identified by immunofluorescence staining of cells ectopically expressed with 53BP1 deletion mutants. This domain consisted of conserved Tudor and Myb motifs. The Tudor plus Myb domain possessed chromatin binding activity in vivo and bound directly to both double-stranded and single-stranded DNA in vitro. This domain also stimulated end-joining by DNA ligase IV/Xrcc4, but not by T4 DNA ligase in vitro. We conclude that 53BP1 has the potential to participate directly in the repair of DNA double-strand breaks
