Evolution of <i>dna</i>Q, the gene encoding the editing 3′ to 5′ exonuclease subunit of DNA polymerase III holoenzyme in Gram‐negative bacteria

Huang, Yiping; Braithwaite, Dan; Ito, Junetsu

doi:10.1016/s0014-5793(96)01361-0

Cited by 19 publications

(23 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…7 Asp (GAC) and 9 Glu (GAA) of blr0640 are amino acid sequence motifs that are conserved among gram-negative bacteria and the exonuclease I motif of the dnaQ gene of E. coli (9,13). Therefore, we constructed a two-base-pair substitution mutant of blr0640 by replacing 7 Asp (GAC) with 7 Ala (GCC), and 9 Glu (GAA) with 9 Ala (GCA) by PCR (Fig.…”

Section: Resultsmentioning

confidence: 99%

Generation of Bradyrhizobium japonicum Mutants with Increased N ₂ O Reductase Activity by Selection after Introduction of a Mutated dnaQ Gene

Itakura

Tabata²,

Eda

et al. 2008

Appl Environ Microbiol

View full text Add to dashboard Cite

We obtained two beneficial mutants of Bradyrhizobium japonicum USDA110 with increased nitrous oxide (N 2 O) reductase (N 2 OR) activity by introducing a plasmid containing a mutated B. japonicum dnaQ gene (pKQ2) and performing enrichment culture under selection pressure for N 2 O respiration. Mutation of dnaQ, which encodes the epsilon subunit of DNA polymerase III, gives a strong mutator phenotype in Escherichia coli. pKQ2 introduction into B. japonicum USDA110 increased the frequency of occurrence of colonies spontaneously resistant to kanamycin. A series of repeated cultivations of USDA110 with and without pKQ2 was conducted in anaerobic conditions under 5% (vol/vol) or 20% (vol/vol) N 2 O atmosphere. At the 10th cultivation cycle, cell populations of USDA110(pKQ2) showed higher N 2 OR activity than the wild-type strains. Four bacterial mutants lacking pKQ2 obtained by plant passage showed 7 to 12 times the N 2 OR activity of the wild-type USDA110. Although two mutants had a weak or null fix phenotype for symbiotic nitrogen fixation, the remaining two (5M09 and 5M14) had the same symbiotic nitrogen fixation ability and heterotrophic growth in culture as wild-type USDA110.

show abstract

Section: Resultsmentioning

confidence: 99%

Generation of Bradyrhizobium japonicum Mutants with Increased N ₂ O Reductase Activity by Selection after Introduction of a Mutated dnaQ Gene

Itakura

Tabata²,

Eda

et al. 2008

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…Genetic studies of the ⑀ subunit of DNA pol III indicate that the HXAXXD sequence in the Exo III⑀ motif defines the active site residues in this region (41,42). The nucleases that contain the HXAXXD sequence include the ⑀ subunits of DNA pols III from Gram-negative bacteria, the exonuclease domains of DNA pols III from Gram-positive bacteria, and the RNases T (38,43,44). The significance of the HXAXXD sequence in the TREX proteins and in the proofreading exonucleases will require additional mutagenesis and structural information on these proteins.…”

Section: Resultsmentioning

confidence: 99%

Identification and Expression of the TREX1 and TREX2 cDNA Sequences Encoding Mammalian 3′→5′ Exonucleases

Mazur

Perrino²

1999

Journal of Biological Chemistry

249

216

View full text Add to dashboard Cite

The 335 exonucleases catalyze the excision of nucleoside monophosphates from the 3 termini of DNA. We have identified the cDNA sequences encoding two 335 exonucleases (TREX1 and TREX2) from mammalian cells. The TREX1 and TREX2 proteins are 304 and 236 amino acids in length, respectively. Analysis of the TREX1 and TREX2 sequences identifies three conserved motifs that likely generate the exonuclease active site in these enzymes. The specific amino acids in these three conserved motifs suggest that these mammalian exonucleases are most closely related to the proofreading exonucleases of the bacterial replicative DNA polymerases and the RNase T enzymes. Expression of TREX1 and TREX2 in Escherichia coli demonstrates that these recombinant proteins are active 335 exonucleases. The recombinant TREX1 protein was purified, and exonuclease activity was measured using single-stranded, partial duplex, and mispaired oligonucleotide DNA substrates. The greatest activity of the TREX1 protein was detected using a partial duplex DNA containing five mispaired nucleotides at the 3 terminus. No activity was detected using single-stranded RNA or an RNA-DNA partial duplex. Identification of the TREX1 and TREX2 cDNA sequences provides the genetic tools to investigate the physiological roles of these exonucleases in mammalian DNA replication, repair, and recombination pathways.The multistep processes of DNA replication, repair, and recombination in human cells often require the excision of nucleotides from the DNA 3Ј termini. For each cell division 4 billion nucleotides must be correctly replicated. The polymerization of incorrect or structurally modified nucleotides into DNA generates the 3Ј termini that block chain elongation by the DNA polymerases. Oxidative damage to DNA can result in fragmented nucleotides at the 3Ј termini that can not be elongated by DNA polymerases. Genetic recombination and mismatch repair pathways can require the removal of normal nucleotides from the 3Ј termini of DNA chains. Enzymes containing 3Ј35Ј exonuclease activity remove these mismatched, modified, fragmented, and normal nucleotides to generate the appropriate 3Ј termini for subsequent steps in the DNA metabolic pathways.Several 3Ј35Ј exonucleases have been described from a variety of animal cells (1-5). These exonucleases demonstrate similar biochemical properties, but the relationships between these enzymes are not known. Also, there are 3Ј35Ј exonucleases contained in the structural domains of mammalian DNA pols 1 ␦ (6), ⑀ (7), and ␥ (8). These proofreading 3Ј35Ј exonucleases excise incorrectly polymerized nucleotides during DNA synthesis. Thus, a variety of 3Ј35Ј exonucleases are present in mammalian cells. These exonucleases might function in multiple pathways to generate 3Ј termini that support further steps such as polymerization or ligation.Excision of incorrectly polymerized nucleotides by proofreading 3Ј35Ј exonucleases is an important mechanism to minimize errors during DNA synthesis. The polymerase-associated proofreading exonuclease was ...

show abstract

“…Divergent evolution could then lead to PolC with its intrinsic exonuclease, and DnaE with its separate proofreading subunit ( ). This divergent evolution step is supported by sequence analysis of exonuclease domains (17).…”

Section: Structural Clues To the Evolution Of The 3 -5 Exonuclease Inmentioning

confidence: 91%

Structure of PolC reveals unique DNA binding and fidelity determinants

Evans

Davies²,

Bullard

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

124

View full text Add to dashboard Cite

PolC is the polymerase responsible for genome duplication in many Gram-positive bacteria and represents an attractive target for antibacterial development. We have determined the 2.4-Å resolution crystal structure of Geobacillus kaustophilus PolC in a ternary complex with DNA and dGTP. The structure reveals nascent base pair interactions that lead to highly accurate nucleotide incorporation. A unique ␤-strand motif in the PolC thumb domain contacts the minor groove, allowing replication errors to be sensed up to 8 nt upstream of the active site. PolC exhibits the potential for large-scale conformational flexibility, which could encompass the catalytic residues. The structure suggests a mechanism by which the active site can communicate with the rest of the replisome to trigger proofreading after nucleotide misincorporation, leading to an integrated model for controlling the dynamic switch between replicative and repair polymerases. This ternary complex of a cellular replicative polymerase affords insights into polymerase fidelity, evolution, and structural diversity.DNA polymerase III ͉ DNA replication ͉ Gram-positive polymerase ͉ polymerase and histidinol phosphatase (PHP) ͉ ternary complex D NA polymerases are the enzymes responsible for DNA synthesis. Cellular organisms typically use multiple DNA polymerase types. The ''replicative'' polymerase performs the bulk of genome duplication, whereas various specialty polymerases repair damaged DNA and resolve Okazaki fragments. Across every kingdom of life, replicative polymerases exhibit certain hallmarks such as high fidelity, speed, and processivity (1). Polymerase holoenzyme accessory proteins play an integral role in achieving the extraordinary efficiency and accuracy of the replicative polymerase complex. These include a ''sliding clamp'' that encircles the DNA and increases processivity (2).Bacterial replicative polymerases comprise the C family of DNA polymerases (3) and differ significantly from the replicative polymerases of eukaryotes, bacteriophage, and archaea, which belong to the B family. The major C family replicative polymerases are DnaE (PolIII), found primarily in Gram-negative bacteria, and PolC (PolIIIC), found primarily in Gram-positive bacteria (4). Apoenzyme crystal structures of DnaE have revealed surprising structural differences in the catalytic center of the enzyme compared with B family polymerases, suggesting a separate evolutionary origin for the C family (5, 6).As the core component of the replicative polymerase complex in Gram-positive pathogens such as Staphylococcus aureus, PolC has received considerable attention as a potential target for antibacterial drug discovery (7,8). No currently marketed antibiotics target the central replication apparatus, making PolC a novel target for antibacterial development. Gram-negative and Gram-positive bacteria are separated by Ͼ1 billion years of evolution, and PolC and DnaE share Ͻ20% sequence identity; PolC is further differentiated from DnaE by domain rearrangements and by the presence of an ...

show abstract

Evolution of dnaQ, the gene encoding the editing 3′ to 5′ exonuclease subunit of DNA polymerase III holoenzyme in Gram‐negative bacteria

Cited by 19 publications

References 30 publications

Generation of Bradyrhizobium japonicum Mutants with Increased N ₂ O Reductase Activity by Selection after Introduction of a Mutated dnaQ Gene

Generation of Bradyrhizobium japonicum Mutants with Increased N ₂ O Reductase Activity by Selection after Introduction of a Mutated dnaQ Gene

Identification and Expression of the TREX1 and TREX2 cDNA Sequences Encoding Mammalian 3′→5′ Exonucleases

Structure of PolC reveals unique DNA binding and fidelity determinants

Contact Info

Product

Resources

About

Evolution of dnaQ, the gene encoding the editing 3′ to 5′ exonuclease subunit of DNA polymerase III holoenzyme in Gram‐negative bacteria

Cited by 19 publications

References 30 publications

Generation of Bradyrhizobium japonicum Mutants with Increased N 2 O Reductase Activity by Selection after Introduction of a Mutated dnaQ Gene

Generation of Bradyrhizobium japonicum Mutants with Increased N 2 O Reductase Activity by Selection after Introduction of a Mutated dnaQ Gene

Identification and Expression of the TREX1 and TREX2 cDNA Sequences Encoding Mammalian 3′→5′ Exonucleases

Structure of PolC reveals unique DNA binding and fidelity determinants

Contact Info

Product

Resources

About

Generation of Bradyrhizobium japonicum Mutants with Increased N ₂ O Reductase Activity by Selection after Introduction of a Mutated dnaQ Gene

Generation of Bradyrhizobium japonicum Mutants with Increased N ₂ O Reductase Activity by Selection after Introduction of a Mutated dnaQ Gene