Neal K. Williams

The unstructured C-terminus of the τ subunit of Escherichia coli DNA polymerase III holoenzyme is the site of interaction with the α subunit

The τ subunit of Escherichia coli DNA polymerase III holoenzyme interacts with the α subunit through its C-terminal Domain V, τC16. We show that the extreme C-terminal region of τC16 constitutes the site of interaction with α. The τC16 domain, but not a derivative of it with a C-terminal deletion of seven residues (τC16Δ7), forms an isolable complex with α. Surface plasmon resonance measurements were used to determine the dissociation constant (KD) of the α−τC16 complex to be ∼260 pM. Competition with immobilized τC16 by τC16 derivatives for binding to α gave values of KD of 7 μM for the α−τC16Δ7 complex. Low-level expression of the genes encoding τC16 and τC16▵7, but not τC16Δ11, is lethal to E. coli. Suppression of this lethal phenotype enabled selection of mutations in the 3′ end of the τC16 gene, that led to defects in α binding. The data suggest that the unstructured C-terminus of τ becomes folded into a helix–loop–helix in its complex with α. An N-terminally extended construct, τC24, was found to bind DNA in a salt-sensitive manner while no binding was observed for τC16, suggesting that the processivity switch of the replisome functionally involves Domain IV of τ.

Mitochondrial Deletions in Normal and Degenerating Rat Retina

Photoreceptor death by apoptosis is the central pathology of most forms of retinal degeneration. Mitochondria play key roles in apoptosis, releasing both signals which induce apoptosis (cytochrome c, caspases) and signals which inhibit apoptosis (Bcl-2). Because mitochondria are the site of oxidative metabolism they are also a major site of formation of the toxic oxygen intermediates which form as oxygen is recruited into the oxidative phosphorylation pathway. Previous studies have shown that deletions in mtDNA accumulate in postmitotic tissues (central nervous, muscle) and that their accumulation is accelerated by oxidative stress (such as hypoxia) (Takeda et al. 1996; Lee et al. 1994; Merril et al. 1996; Englander et al. 1999). It seems possible therefore that mitochondria are a site at which oxidative stress induces the death of retinal neurones. This study investigates the accumulation of mtDNA deletions in the rat retina, in both normal (non-degenerative) and degenerative strains. Deletions were undetectable in Sprague-Dawley albino rats (24 months) but were detected at 15 months in the rapidly degenerating RCS strain. The appearance of deletions in the RCS strain, in which retinal oxygen tension is known to rise as the degeneration progresses, gives support to the ideas that oxidative stress is a factor in mtDNA deletions, and in the progress of the late stages of the degeneration.

Homology and Mutagenesis Studies of Hamster Dihydroorotase

The third reaction in de novo pyrimidine biosynthesis is catalyzed by dihydroorotase (for details see Williams et al., in this volume). By screening a variety of structural analogues of N-carbamyl-L-aspartate (CA-asp) and L-dihydroorotate (DHO), Christopherson and Jones (1980) found dihydroorotase to be highly specific for its natural substrates. Orotate and 5-substituted derivatives, such as 5-fluoroorotate, are effective inhibitors, but dihydrouracil and the CA-asp analogues, N-carbamyl-β-alanine, N-carbamyl-α-alanine, and N-acetyl-L-aspartate are not inhibitory. These observations suggest the identity of essential attachment points in the enzyme-substrate complex. Dihydrouracil lacks the carboxylate group at position 4 of dihydroorotase, and N-carbamyl-β-alanine lacks the corresponding α-carboxylate of CA-asp demonstrating that this group is required for substrate binding, possibly by interacting with a positively charged enzymic group (Christopherson and Jones, 1980). N-carbamyl-a-alanine is lacking the β-carboxylate of CA-asp and N-acetyl-L-aspartate does not possess the terminal ureido nitrogen of the substrate, indicating attachments at these locations. The β-carboxylate of CA-asp may form a coordination complex with the active site zinc atom (see Williams et al., in this volume). We have used knowledge of the cDNA sequence of hamster dihydroorotase in combination with site-directed mutagenesis in an attempt to identify the amino acids involved in these substrate attachments.

The Catalytic Mechanism of Hamster Dihydroorotase

Dihydroorotase catalyses the third reaction of the pathway for de novo biosynthesis of pyrimidine nucleotides. The reaction catalysed involves an intramolecular ring closure of N-carbamyl-L-aspartate (CA-asp) to form L-dihydroorotate (DHO), a dihydropyrimidine. The interconversion of CA-asp and DHO is freely reversible (Christopherson and Jones, 1979) and the pH-rate profiles for the biosynthetic and degradative reactions and inhibition of the enzyme by L-cysteine suggested that mammalian dihydroorotase has a catalytic mechanism similar to carboxypeptidase A (Christopherson and Jones, 1980) with a zinc atom at the active site. This zinc atom would polarise the =C=O group of the scissile peptide bond in the dihydropyrimidine ring of DHO and stabilise the tetrahedral, dioxy anionic transition state of the reaction (Fig. 1). Taylor et al. (1976) had purified the dihydroorotase from Clostridium oroticum and found two gram atoms of zinc per subunit and similar pH-rate profiles. Kelly et al. (1986) subsequently demonstrated that hamster dihydroorotase also contained an atom of zinc which was required for catalysis. It therefore seems likely that mammalian dihydroorotase does have a catalytic mechanism which resembles that of zinc protease.