Arthur E. Pritchard

A novel assembly mechanism for the DNA polymerase III holoenzyme DnaX complex: association of δδ′ with DnaX4 forms DnaX3δδ′

We have constructed a plasmid‐borne artificial operon that expresses the six subunits of the DnaX complex of Escherichia coli DNA polymerase III holoenzyme: τ, γ, δ, δ′, χ and ψ. Induction of this operon followed by assembly in vivo produced two τγ mixed DnaX complexes with stoichiometries of τ1γ2δδ′χψ and τ2γ1δδ′χψ rather than the expected γ2τ2δδ′χψ. We observed the same heterogeneity when τγ mixed DnaX complexes were reconstituted in vitro. Re‐examination of homomeric DnaX τ and γ complexes assembled either in vitro or in vivo also revealed a stoichiometry of DnaX3δδ′χψ. Equilibrium sedimentation analysis showed that free DnaX is a tetramer in equilibrium with a free monomer. An assembly mechanism, in which the association of heterologous subunits with a homomeric complex alters the stoichiometry of the homomeric assembly, is without precedent. The significance of our findings to the architecture of the holoenzyme and the clamp‐assembly apparatus of all other organisms is discussed.

τ Binds and Organizes Escherichia coli Replication Proteins through Distinct Domains DOMAIN III, SHARED BY γ AND τ, OLIGOMERIZES DnaX

The τ and γ proteins of the DNA polymerase III holoenzyme DnaX complex are products of the dnaX gene with γ being a truncated version of τ arising from ribosomal frameshifting. τ is comprised of five structural domains, the first three of which are shared by γ (Gao, D., and McHenry, C. (2001)J. Biol. Chem. 276, 4433–4453). In the absence of the other holoenzyme subunits, DnaX exists as a tetramer. Association of δ, δ′, χ, and ψ with domain III of DnaX4 results in a DnaX complex with a stoichiometry of DnaX3δδ′χψ. To identify which domain facilitates DnaX self-association, we examined the properties of purified biotin-tagged DnaX fusion proteins containing domains I-II or III-V. Unlike domain I-II, treatment of domain III-V, γ, and τ with the chemical cross-linking reagent BS3 resulted in the appearance of high molecular weight intramolecular cross-linked protein. Gel filtration of domains I-II and III-V demonstrated that domain I-II was monomeric, and domain III-V was an oligomer. Biotin-tagged domain III-V, and not domain I-II, was able to form a mixed DnaX complex by recruiting τ, δ, δ′, χ, and ψ onto streptavidin-agarose beads. Thus, domain III not only contains the δ, δ′, χ, and ψ binding interface, but also the region that enables DnaX to oligomerize.

An unusual region of Paramedum mitochondrial DNA containing chloroplast-like genes

Based on DNA and amino acid comparisons with known genes and their products, a region of the Paramecium aurelia mitochondrial (mt) genome has been found to encode the following gene products: (1) photosystem II protein G (psbG); (2) a large open reading frame (ORF400) which is also found encoded in the chloroplast (cp) DNA of tobacco (as ORF393) and liverwort (as ORF392), and in the kinetoplast maxicircle DNA of Leishmania tarentolae (as ORFs 3 and 4); (3) ribosomal protein L2 (rpl2); (4) ribosomal protein S12 (rps12); (5) ribosomal protein S14 (rps14); and (6) NADH dehydrogenase subunit 2 (ndh2). All of these genes have been found in cp DNA, but the psbG gene has never been identified in a mt genome, and ribosomal protein genes have never been located in an animal or protozoan mitochondrion. The ndh2 gene has been found in both mitochondria and plastids. The Paramecium genes are among the most divergent of those sequenced to date. Two of the genes are encoded on the strand of DNA complementary to that encoding all other known Paramecium mt genes. No gene contains an identifiable intron. The rps12 and psbG genes are probably overlapping. It is not yet known whether these genes are transcribed or have functional gene products. The presence of these genes in the mt genome raises interesting questions concerning their evolutionary origin.

A Three-domain Structure for the δ Subunit of the DNA Polymerase III Holoenzyme δ Domain III Binds δ′ and Assembles into the DnaX Complex

Using ψ-BLAST, we have developed a method for identifying the poorly conserved δ subunit of the DNA polymerase III holoenzyme from all sequenced bacteria. This approach, starting withEscherichia coli δ, leads not only to the identification of δ but also to the DnaX and δ′ subunits of the DnaX complex and other AAA+-class ATPases. This suggests that, although not an ATPase, δ is related structurally to the other subunits of the DnaX complex that loads the β sliding clamp processivity factor onto DNA. To test this prediction, we aligned δ sequences with those of δ′ and, using the start of δ′ Domain III established from its x-ray crystal structure, predicted the juncture between Domains II and III of δ. This putative δ Domain III could be expressed to high levels, consistent with the prediction that it folds independently. δ Domain III, like Domain III of DnaX and δ′, assembles by itself into a complex with the other DnaX complex components. Cross-linking studies indicated a contact of δ with the DnaX subunits. These observations are consistent with a model where two τ subunits and one each of the γ, δ′, and δ subunits mutually interact to form a pentameric functional core for the DnaX complex.

Replication Mechanism of Mitochondrial DNA from Paramecium aurelia: Sequence of the Cross-linked Origin

With all the elegant genetic and biochemical information on fungal, plant, and mammalian mitochondria presented in this volume, one might ask, Why study Paramecium ? In many respects, Paramecium is a choice organism. Its size (150 μ m × 100 μ m) makes it easily amenable to microinjection, and it was in this organism that transformation to antibiotic resistance by the direct injection of purified mitochondria was first demonstrated (see Beale and Knowles 1976). Moreover, by examination of mtDNAs from donor, recipient, and transformed cells, we were able to demonstrate that it was indeed the donor DNA that brought about this transformation (Cummings et al. 1976). In addition, Paramecium mtDNA may represent an intermediate case between yeast and mammalian cells: GC content, 37%; size, 14 μ m; lack of intervening sequences in rDNA; and a separation of rRNA genes of about 10 kbp (Maki and Cummings 1977; Cummings et al. 1980). But most importantly, mtDNA from Paramecium is a linear molecule (Goddard and Cummings 1975), making it unlike other mtDNAs. Only in Tetrahymena, another ciliate, has linear mtDNA also been demonstrated (Suyama and Miura 1968). On the basis of electron microscopy studies, the replication scheme of the linear mtDNA from Paramecium can be depicted as in Figure 1. Partial denaturation studies showed that there is an AT-rich “bubble” at one end of the monomer molecule, and this is shown here as a cross-link. Replication proceeds by means of “lariat” intermediates, terminating in a dimer-length linear molecule. Growth in the presence of ethidium bromide caused...