Affinity purification of antibodies by using Ni2+-resins on which inclusion body-forming proteins are immobilized.

Bacterially expressed recombinant proteins are widely used for producing specific antibodies. Unfortunately, many recombinant proteins are recovered as insoluble materials, so-called inclusion bodies. Inclusion bodies are rather advantageous from a point of view of immunogens because fairly pure proteins can be feasibly extracted from the inclusion bodies. However, we encounter a problem with an insoluble protein when we make an antigen-immobilized column for affinity purification of antibodies because we need a soluble protein in usual immobilization methods. Histidine-tagged proteins can be bound to Ni(2+)-resins in buffer containing 6M guanidine-HCl, in which most insoluble proteins are solubilized. Taking advantage of this feature, we have successfully purified antigen-specific antibodies by directly using Ni(2+)-resins onto which denatured proteins are bound.

Ditercalinium chloride, a pro-anticancer drug, intimately associates with mammalian mitochondrial DNA and inhibits its replication.

Ditercalinium chloride was originally synthesized for use as an anticancer drug and was then found to deplete mitochondrial DNA. Ethidium bromide is widely used to deplete mitochondrial DNA and produce mitochondrial DNA-less cell lines. Although ethidium bromide is used in the case of human cell lines, it frequently fails to deplete mitochondrial DNA in mouse cells. In contrast, ditercalinium chloride can deplete mitochondrial DNA in both mouse and human cells. However, little is known of the mechanisms by which ditercalinium chloride depletes mitochondrial DNA. Here, we show that ditercalinium chloride inhibits human DNA polymerase gamma activity as efficiently as does ethidium bromide. Ethidium bromide accumulates much less in mouse B82 cells, as compared with findings in human HeLa cells, whereas ditercalinium chloride accumulates in both to a similar extent. This poor accumulation of ethidium bromide may, in part, account for the resistance. Ethidium bromide distributes diffusely in the mitochondria of HeLa cells, while ditercalinium chloride distributes granularly and hence may be strongly associated with mitochondrial DNA. Each granular spot presumably represents one mitochondrial DNA nucleoid. In support of this idea, ditercalinium chloride co-localizes with Twinkle, a mitochondrial helicase and is assumed to associate with mitochondrial DNA. This close association of ditercalinium chloride with mitochondrial DNA may contribute to the mitochondrial DNA-depleting activity.

Mammalian mitochondrial endonuclease G. Digestion of R-loops and localization in intermembrane space.

Mammalian mitochondria contain strong nuclease activity. Endonuclease G (endoG), which predominantly resides in mitochondria, accounts for a large part of this nuclease activity. It has been proposed to act as an RNase H-like nuclease on RNA.DNA hybrids (R-loops) in the D-loop region where the origins of mitochondrial replication are mapped, providing RNA primers for mtDNA replication. However, in contrast with this proposed activity, endoG has recently been shown to translocate to nuclei on apoptotic stimulation and act as a nuclease without sequence specificity. To clarify the role of endoG in mtDNA replication, we examined its submitochondrial localization and its ability to cleave R-loops. At low concentration, it preferentially produces double-stranded breaks in R-loops, but does not act as an RNase H-like nuclease. In addition, it exists in the mitochondrial intermembrane space, but not in the matrix where mtDNA replication occurs. These results do not support the involvement of endoG in mtDNA replication. Based on the fact that guanine tracts, which are preferential targets of endoG, tend to form triplex structures and that endoG produces double-stranded breaks in R-loops, we propose that three-stranded DNA may be the preferred substrate of endoG.

1-Methyl-4-phenylpyridinium ion, a toxin that can cause parkinsonism, alters branched structures of DNA.

During replication, human mitochondrial DNA (mtDNA) takes on a triple-stranded structure known as a D-loop, which is implicated in replication and transcription. 1-Methyl-4-phenylpyridinium ion (MPP+), a toxin inducing parkinsonism, inhibits mtDNA replication, possibly by resolving the D-loops. For initiation of mtDNA replication, mitochondria are thought to have another triple-stranded structure, an R-loop. The R-loop, which is resolved by a bacterial junction-specific helicase, RecG, is also resolved by MPP+. Because mitochondrial D-loops are likewise resolved by RecG, the D- and R-loops may share a similar branched structure. MPP+ resolves cruciform DNA in supercoiled DNA. MPP+ converts a stacked conformation to an extended conformation in a synthetic Holliday junction. This conversion is reversed by 1 mM Mg(2+), as is the resolution of the D-loops or cruciform DNA. These observations suggest that the junction structure of mitochondrial D- and R-loops is affected by MPP+.

Regulation of mitochondrial D-loops by transcription factor A and single-stranded DNA-binding protein.

During replication, mitochondrial DNA (mtDNA) takes on a triple-stranded structure called a D-loop. Although their physiological roles are not understood, D-loops are implicated in replication and transcription of mtDNA. Little is known about the turnover of D-loops. We investigated the effects of mitochondrial transcription factor A (TFAM) and single-stranded DNA-binding protein (mtSSB) on D-loops. In human HeLa cells, TFAM and mtSSB are, respectively, 1700- and 3000-fold more abundant than mtDNA. This level of TFAM is two orders of magnitude higher than reported previously and is sufficient to wrap human mtDNA entirely. TFAM resolves D-loops in vitro if added in similar stoichiometries. mtSSB inhibits the resolution of mtDNA by TFAM but enhances resolution by RecG, a junction-specific helicase from Escherichia coli. Hence, mtSSB functions in both stabilization and resolution. We propose that TFAM and mtSSB are cooperatively involved in stabilizing D-loops and in the maintenance of mtDNA.