Calf thymus Hsc70 and Hsc40 can substitute for DnaK and DnaJ function in protein renaturation but not in bacteriophage DNA replication.

Calf thymus (ct) Hsc70 has been shown previously to reactivate heat-inactivated prokaryotic and eukaryotic enzymes, while DnaK was able to reactivate solely prokaryotic enzymes. Here, we report on isolation from calf thymus of a DnaJ homolog, ctHsc40, and on testing of its cooperative function in three different assays: (i) reactivation of heat-inactivated DNA polymerases, (ii) stimulation of the ATPase activity of ctHsc70 chaperone, and (iii) replication of bacteriophage lambda DNA. Surprisingly, ctHsc70/ctHsc40 chaperones were found to reactivate the denatured prokaryotic and eukaryotic enzymes but not to promote bacteriophage lambda DNA replication, suggesting species specificity in DNA replication.

Import of Agrobacterium T-DNA into plant nuclei: two distinct functions of VirD2 and VirE2 proteins.

To study the mechanism of nuclear import of T-DNA, complexes consisting of the virulence proteins VirD2 and VirE2 as well as single-stranded DNA (ssDNA) were tested for import into plant nuclei in vitro. Import of these complexes was fast and efficient and could be inhibited by a competitor, a nuclear localization signal (NLS) coupled to BSA. For import of short ssDNA, VirD2 was sufficient, whereas import of long ssDNA additionally required VirE2. A VirD2 mutant lacking its C-terminal NLS was unable to mediate import of the T-DNA complexes into nuclei. Although free VirE2 molecules were imported into nuclei, once bound to ssDNA they were not imported, implying that when complexed to DNA, the NLSs of VirE2 are not exposed and thus do not function. RecA, another ssDNA binding protein, could substitute for VirE2 in the nuclear import of T-DNA but not in earlier events of T-DNA transfer to plant cells. We propose that VirD2 directs the T-DNA complex to the nuclear pore, whereas both proteins mediate its passage through the pore. Therefore, by binding to ssDNA, VirE2 may shape the T-DNA complex such that it is accepted for translocation into the nucleus.

Odyssey of agrobacterium T-DNA.

Agrobacterium tumefaciens, a plant pathogen, is characterized by the unique feature of interkingdom DNA transfer. This soil bacterium is able to transfer a fragment of its DNA, called T-DNA (transferred DNA), to the plant cell where T-DNA is integrated into the plant genome leading to "genetic colonization" of the host. The fate of T-DNA, its processing, transfer and integration, resembles the journey of Odysseus, although our hero returns from its long trip in a slightly modified form.

Plant enzymes but not Agrobacterium VirD2 mediate T-DNA ligation in vitro.

Agrobacterium tumefaciens, a gram-negative soil bacterium, transfers DNA to many plant species. In the plant cell, the transferred DNA (T-DNA) is integrated into the genome. An in vitro ligation-integration assay has been designed to investigate the mechanism of T-DNA ligation and the factors involved in this process. The VirD2 protein, which is produced in Agrobacterium and is covalently attached to T-DNA, did not, under our assay conditions, ligate T-DNA to a model target sequence in vitro. We tested whether plant extracts could ligate T-DNA to target oligonucleotides in our test system. The in vitro ligation-integration reaction did indeed take place in the presence of plant extracts. This reaction was inhibited by dTTP, indicating involvement of a plant DNA ligase. We found that prokaryotic DNA ligases could substitute for plant extracts in this reaction. Ligation of the VirD2-bound oligonucleotide to the target sequence mediated by T4 DNA ligase was less efficient than ligation of a free oligonucleotide to the target. T-DNA ligation mediated by a plant enzyme(s) or T4 DNA ligase requires ATP.

Import of DNA into mammalian nuclei by proteins originating from a plant pathogenic bacterium.

Import of DNA into mammalian nuclei is generally inefficient. Therefore, one of the current challenges in human gene therapy is the development of efficient DNA delivery systems. Here we tested whether bacterial proteins could be used to target DNA to mammalian cells. Agrobacterium tumefaciens, a plant pathogen, efficiently transfers DNA as a nucleoprotein complex to plant cells. Agrobacterium-mediated T-DNA transfer to plant cells is the only known example for interkingdom DNA transfer and is widely used for plant transformation. Agrobacterium virulence proteins VirD2 and VirE2 perform important functions in this process. We reconstituted complexes consisting of the bacterial virulence proteins VirD2, VirE2, and single-stranded DNA (ssDNA) in vitro. These complexes were tested for import into HeLa cell nuclei. Import of ssDNA required both VirD2 and VirE2 proteins. A VirD2 mutant lacking its C-terminal nuclear localization signal was deficient in import of the ssDNA-protein complexes into nuclei. Import of VirD2-ssDNA-VirE2 complexes was fast and efficient, and was shown to depended on importin alpha, Ran, and an energy source. We report here that the bacterium-derived and plant-adapted protein-DNA complex, made in vitro, can be efficiently imported into mammalian nuclei following the classical importin-dependent nuclear import pathway. This demonstrates the potential of our approach to enhance gene transfer to animal cells.

Calf thymus Hsc70 protein protects and reactivates prokaryotic and eukaryotic enzymes.

The heat-shock 70 protein (Hsp70) chaperone family is very conserved and its prokaryotic homologue, the DnaK protein, is assumed to form one of the cellular systems for the prevention and restoration of heat-induced protein denaturation. By using anti-DnaK antibodies we purified the DnaK homologue heat-shock cognate protein (Hsc70) from calf thymus to apparent homogeneity. This protein was classified as an eukaryotic Hsc70, since (i) monoclonal antibodies against eukaryotic Hsc70 recognized it, (ii) its amino-terminal sequence showed strong homology to Hsp70s from eukaryotes and, (iii) it had an intrinsic weak ATPase activity that was stimulated by various peptide substrates. We show that this calf thymus Hsc70 protein protected calf thymus DNA polymerases alpha and epsilon as well as Escherichia coli DNA polymerase III and RNA polymerase from heat inactivation and could reactivate these heat-inactivated enzymes in an ATP-hydrolysis dependent manner, likely leading to the dissociation of aggregates formed during heat inactivation. In contrast to this, DnaK protein was exclusively able to protect and to reactivate the enzymes from E.coli but not from eukaryotic cells. Finally, the addition of calf thymus DnaJ co-chaperone homologue reduced the amount of Hsc70 required for reactivation at least 10-fold.

Both the Escherichia coli chaperone systems, GroEL/GroES and DnaK/DnaJ/GrpE, can reactivate heat-treated RNA polymerase. Different mechanisms for the same activity.

In this work we show that the GroEL (Hsp60 equivalent) chaperone protein can protected purified Escherichia coli RNA polymerase (RNAP) holoenzyme from heat inactivation better than the DnaK (Hsp70 equivalent) chaperone can. In this protection reaction, the GroES protein is not essential, but its presence reduces the amount of GroEL required. GroEL and GroES can also reactivate heat-inactivated RNAP in the presence of ATP. The mutant GroEL673 protein, with or without GroES, is incapable of reactivating heat-inactivated RNAP. GroEL673 can only protect RNAP, and this protecting ability is not stimulated by GroES. The mechanism by which the DnaJ and GrpE heat shock proteins contribute to DnaK's ability to reactivate heat-inactivated RNAP GroEL673 has also been investigated. We found that the DnaJ protein substantially reduces the levels of DnaK protein needed in this reactivation assay. However, the observed lag in reactivation is diminished only in the additional presence of the GrpE protein. Hence, DnaJ and GrpE are involved in both steps of this reactivation reaction (recognition of substrate and release of chaperone from the substrate-chaperone complex) while, in the case of the GroEL-dependent reaction, GroES is involved only during the release of chaperone from the substrate-chaperone complex.