Dysfunctional proofreading in the <i>Escherichia coli</i> DNA polymerase III core

Lehtinen, Duane A.; Perrino, Fred W.

doi:10.1042/bj20040660

Cited by 13 publications

(12 citation statements)

References 54 publications

(105 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A functional co-dependence of the polymerase and exonuclease activities of Pol III ␣ and ⑀ subunits has been observed previously (22,23). Here, we note a similar relationship between the polymerase activity and the php ⑀-binding domain of ␣.…”

Section: Discussionsupporting

confidence: 89%

“…Binding of ⑀ to ␣ stimulates polymerase activity 2-fold and proofreading activity 10 -80-fold, depending on the substrate (22). Studies with mutant ⑀ subunits suggest that a functional interaction is required in addition to tight tethering for functional coordination of synthesis with proofreading (23), and the existence of ⑀ and ␣ as separate polypeptide chains provides a convenient experimental system for studying this integration. The usefulness of such an experimental system requires identification of the sites of subunit interaction.…”

mentioning

confidence: 99%

See 1 more Smart Citation

The NH2-terminal php Domain of the α Subunit of the Escherichia coli Replicase Binds the ϵ Proofreading Subunit

Wieczorek

McHenry

2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

The ␣ subunit of the replicase of all bacteria contains a php domain, initially identified by its similarity to histidinol phosphatase but of otherwise unknown function (Aravind, L., and Koonin, E. V. (1998) Nucleic Acids Res. 26, 3746 -3752). Deletion of 60 residues from the NH 2 terminus of the ␣ php domain destroys ⑀ binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for ⑀ binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds ⑀ with the same affinity as the 1160-residue full-length ␣ subunit. A subset of mutations of a conserved acidic residue (Asp 43 in Escherichia coli ␣) present in the php domain of all bacterial replicases resulted in defects in ⑀ binding. Using sequence alignments, we show that the prototypical Gram(؉) Pol C, which contains the polymerase and proofreading activities within the same polypeptide chain, has an ⑀-like sequence inserted in a surface loop near the center of the homologous YcdX protein. These findings suggest that the php domain serves as a platform to enable coordination of proofreading and polymerase activities during chromosomal replication.The initial discovery of a proofreading 3Ј35Ј exonuclease within DNA polymerase I provided the prototype for proofreading among all polymerases (1). Editing activity was found in the same polypeptide chain as Pol 2 I and required exchange of the 3Ј-primer terminus between the polymerase and proofreading exonuclease sites, which are separated by ϳ30 Å (2). The structure of the exonuclease site and functional studies with cross-linked substrates suggested that ϳ4 bases needed to be unwound to permit migration of the 3Ј end of a primer from the Pol to exonuclease active site (2, 3). This provided a basis for the preference of the 3Ј35Ј exonuclease for mispaired over properly base-paired primer termini. Partitioning of the primer terminus between the exonuclease and polymerase sites is determined by both DNA structure and protein contacts (4). The kinetic basis for efficient proofreading is the very slow rate of elongation of primers containing a terminal mismatch, providing time for partitioning (5, 6). The exonucleolytic mechanism requires two Mg 2ϩ ions tethered by acidic side chains within the polymerase active site (2), which demonstrates the close functional relationship between the two activities. Escherichia coli Pol II also contains an exonuclease activity in the same polypeptide chain as the polymerase (7).The multisubunit core of the E. coli Pol III replicase was found to contain the polymerase subunit ␣ bound to subunits ⑀ and (8). ⑀ is the product of the mutD gene and was initially identified as conferring high levels of spontaneous mutagenesis when defective (9). In contrast to Pol I and II, ⑀ was shown to be the separate proofreading subunit of the Pol III replicase (10 -13). Holoenzyme reconstituted with ⑀ also exhibits lower fidelity in vitro (11,14).Similar proofreading activities are found in eukaryotic nu...

show abstract

Section: Discussionsupporting

confidence: 89%

mentioning

confidence: 99%

The NH2-terminal php Domain of the α Subunit of the Escherichia coli Replicase Binds the ϵ Proofreading Subunit

Wieczorek

McHenry

2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…An increase of 1000-fold in the mutability of a dnaQ49 (V96G) strain was observed in the absence of (9). The subunit also stimulates the exonuclease activity of the ⑀ double mutant I170T/V215A when present in the Pol III core complex (10). This observation supports a role for in coordinating the ␣-⑀ polymerase-exonuclease interaction (10).…”

mentioning

confidence: 61%

Structure of the Escherichia coli DNA Polymerase III ϵ-HOT Proofreading Complex

Kirby

Harvey

DeRose

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

The ⑀ subunit of Escherichia coli DNA polymerase III possesses 3-exonucleolytic proofreading activity. Within the Pol III core, ⑀ is tightly bound between the ␣ subunit (DNA polymerase) and subunit. Here, we present the crystal structure of ⑀ in complex with HOT, the bacteriophage P1-encoded homolog of , at 2.1 Å resolution. The ⑀-HOT interface is defined by two areas of contact: an interaction of the previously unstructured N terminus of HOT with an edge of the ⑀ central ␤-sheet as well as interactions between HOT and the catalytically important helix ␣1-loop-helix ␣2 motif of ⑀. This structure provides insight into how HOT and, by implication, may stabilize the ⑀ subunit, thus promoting efficient proofreading during chromosomal replication.The precise mechanisms by which cells are able to duplicate their DNA with both high accuracy (Ͻ1 error/10 10 bases replicated) and speed (up to 1000 nucleotides/s) are of major interest. Chromosomal replication is performed by multisubunit replicases that conduct the simultaneous, coordinated replication of the leading and lagging strands. Among the model systems currently being investigated, the best understood is that of the bacterium Escherichia coli (1, 2). In this organism, chromosomal replication is performed by DNA polymerase III holoenzyme (HE) 2 , a dimeric complex containing 10 distinct subunits (17 total). Within the HE complex (␣⑀) 2 ␤ 4 2 (␥␦␦Ј), there are two polymerase core assemblies (␣⑀), one for each strand, which are the primary determinants of replication fidelity. Each core consists of the ␣ subunit (the polymerase, (M r ϭ 135,000)), the ⑀ subunit (a 3Ј 3 5Ј exonuclease that acts as a proofreader for polymerase misinsertion errors, (M r ϭ 28,500)), and the subunit (M r ϭ 8,000), connected in the linear order ␣-⑀-.The ⑀ subunit, encoded by the dnaQ gene, plays a critical role within the Pol III core, both catalytically and structurally. Many dnaQ mutants exhibit strong mutator phenotypes, whereas a fully catalytically deficient mutation causes lethality due to excessively high mutation rates (error catastrophe) (3). Deletion mutants of dnaQ have been generated, but they also proved to be nonviable unless accompanied by a suppressing mutation in the polymerase (4). Based on these studies, ⑀ is thought to have at least two functions: a fidelity function and a structural function, due to its tight and presumably stabilizing interaction with the polymerase. Recently, a potential second proofreading activity has been discovered residing in the N-terminal PHP domain of the ␣ subunit (5, 6), which also contains the binding site for ⑀. Interaction between ⑀ and ␣ is dependent on the C-terminal domain of ⑀ (residues 187-243) (7,8). In contrast, the N-terminal domain of ⑀ (residues 1-186) contains the exonuclease active site and retains binding affinity for the subunit.The subunit does not have any known enzymatic function; its role within the Pol III core is presumed to be structural, through its interaction with the ⑀ subunit. Strains lacking (⌬holE mutants), al...

show abstract

“…On the other hand, experimental results have reported that the addition of θ to ε have little measurable effect on the exonuclease activity 10. Also, free– ε 186 has been shown to have a slightly lower energy barrier (0.2 kcal/mol) than ε 186– θ with pNP–TMP and Mn 2+ 8.…”

Section: Resultsmentioning

confidence: 98%

“…Association of ε with θ has been proposed to promote exonuclease activity 9,10. In addition, it has recently been shown that a homolog of theta (HOT) from bacteriophage P1 can substitute for θ and has been proposed to stabilize ε 11,12.…”

Section: Introductionmentioning

confidence: 99%

Reaction Mechanism of the ε Subunit of E. coli DNA Polymerase III: Insights into Active Site Metal Coordination and Catalytically Significant Residues

et al. 2009

View full text Add to dashboard Cite

The 28kDa ε subunit of Escherichia coli DNA polymerase III is the exonucleotidic proofreader responsible for editing polymerase insertion errors. Here, we study the mechanism by which ε carries out the exonuclease activity. We performed quantum mechanics/molecular mechanics calculations on the N–terminal domain containing the exonuclease activity. Both the free–ε and a complex, ε bound to a θ homolog (HOT), were studied. For the ε–HOT complex, Mg2+ or Mn2+ were investigated as the essential divalent metal cofactors, while only Mg2+ was used for free–ε. In all calculations, a water molecule bound to the catalytic metal acts as the nucleophile for the hydrolysis of the phosphate bond. Initially, a direct proton transfer to H162 is observed. Subsequently, the nucleophilic attack takes place, followed by a second proton transfer to E14. Our results show that the reaction catalyzed with Mn2+ is faster than with Mg2+, in agreement with experiment. In addition, the ε–HOT complex shows a slightly lower energy barrier compared to free–ε. In all cases the catalytic metal is observed to be penta–coordinated. Charge and frontier orbital analyses suggest that charge transfer may stabilize the penta–coordination. Energy decomposition analysis to study the contribution of each residue to catalysis suggests that there are several important residues. Among these, H98, D103, D129 and D146 have been implicated in catalysis by mutagenesis studies. Some of these residues were found to be structurally conserved on human TREX1, the exonuclease domains from E. coli DNA–Pol I, and the DNA polymerase of bacteriophage RB69.

show abstract

Dysfunctional proofreading in the Escherichia coli DNA polymerase III core

Cited by 13 publications

References 54 publications

The NH2-terminal php Domain of the α Subunit of the Escherichia coli Replicase Binds the ϵ Proofreading Subunit

The NH2-terminal php Domain of the α Subunit of the Escherichia coli Replicase Binds the ϵ Proofreading Subunit

Structure of the Escherichia coli DNA Polymerase III ϵ-HOT Proofreading Complex

Reaction Mechanism of the ε Subunit of E. coli DNA Polymerase III: Insights into Active Site Metal Coordination and Catalytically Significant Residues

Contact Info

Product

Resources

About