Structural Basis for Proofreading during Replication of the Escherichia coli Chromosome

Abstract

The epsilon subunit of the Escherichia coli replicative DNA polymerase III is the proofreading 3'-5' exonuclease. Structures of its catalytic N-terminal domain (epsilon186) were determined at two pH values (5.8 and 8.5) at resolutions of 1.7-1.8 A, in complex with two Mn(II) ions and a nucleotide product of its reaction, thymidine 5'-monophosphate. The protein structure is built around a core five-stranded beta sheet that is a common feature of members of the DnaQ superfamily. The structures were identical, except for differences in the way TMP and water molecules are coordinated to the binuclear metal center in the active site. These data are used to develop a mechanism for epsilon and to produce a plausible model of the complex of epsilon186 with DNA.

QIP, a putative exonuclease, interacts with the Neurospora Argonaute protein and facilitates conversion of duplex siRNA into single strands

“…Furthermore, secondary structure alignment ( Fig. 2D) showed that the predicted QIP exonuclease domain was remarkably similar to that in the Escherichia coli DNA polymerase III subunit, for which a crystal structure has been solved (Hamdan et al 2002). Together, these alignment studies suggest that QIP possesses a 3Ј-5Ј DEDDh exonuclease domain.…”

“…Comparison with various 3Ј-5Ј exonuclease domains suggests that the exonuclease domain in QIP belongs to the DEDDh superfamily of 3Ј-5Ј exonucleases. Figure 2C shows the amino acid alignment of three critical motifs of various 3Ј-5Ј exonuclease domains in QIP and other proteins (Hamdan et al 2002). The three motifs have four invariant acidic residues (D and E in motif I, D in motif II, and H and D in motif III) in the 3Ј-5Ј exonuclease domains; all are conserved in QIP.…”

“…(D) Secondary structure comparison between the exonuclease domains in QIP and DnaQ (E. coli DNA polymerase III subunit). The predicted secondary structure of QIP was obtained by JPred (http://www.compbio.dundee.ac.uk/∼www-jpred), whereas the secondary structure of DnaQ was based on its crystal structure (Hamdan et al 2002). in motif III functions as a general base to direct an activated H 2 O molecule for nucleophilic attack at the terminal phosphodiester bond and is critical for exonuclease activity (Hamdan et al 2002). The known RNAi suppressor ERI-1 also contains an exonuclease domain (Kennedy et al 2004).…”

Discovery of an RNA virus 3′→5′ exoribonuclease that is critically involved in coronavirus RNA synthesis

“…The data presented in Figs. 6 and 7B, together with sequence comparisons of CoV ExoN domains with DEDD family exonucleases (13), and the biochemical and structural information available for several of these cellular homologs (24)(25)(26)(27)(28)(29), suggest that the ExoN activity critically depends on two divalent metal ions that are coordinated by the SARS-CoV pp1ab residues Asp-5992 and Glu-5994 (both from Exo I), Asp-6145 (Exo II), and Asp-6175 (Exo III). With few exceptions (30), the conserved Exo I-III Asp and Glu residues are known to be essential for DEDD nuclease activity, whereas replacements of the conserved Exo III Tyr͞His residue appear to be slightly less detrimental (26,31,32).…”

“…Given that the various DEDD family members use a similar but not identical set of residues to coordinate the two metal ions and to orient the attacking hydroxide ion and its nucleotide substrate (24,25,27,30,33), more experimentation and structure information will be required to understand the precise functional and structural role of individual ExoN residues and the other subdomains present in nidovirus nsp14 proteins. In this context, the role of the putative Zn-finger structure, which uniquely separates the Exo I and II motifs in CoV and torovirus ExoNs (13), will be of major interest.…”