Role of <i>Escherichia coli</i> DNA polymerase I in chromosomal DNA replication fidelity

Makiela‐Dzbenska, Karolina; Jaszczur, Malgorzata; Banach-Orlowska, Magdalena; Jonczyk, Piotr; Schaaper, Roel M.; Fijałkowska, Iwona J.

doi:10.1111/j.1365-2958.2009.06921.x

Cited by 32 publications

(35 citation statements)

References 76 publications

(121 reference statements)

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“…Inactivation of the Pol I 3′ exonuclease causes a modest, 2–4 fold increase in specific lacZ reversion rates. This is apparent in an orientation-specific manner, confirming the expectation that Pol I errors arise primarily on the lagging strand (177). In this study, deficiency in the 3′ exonuclease of Pol I did not strongly enhance the mutator phenotype conferred by defects in the Polymerase III holoenzyme, suggesting that Pol I does not readily replace Pol III in the processing of misincorporation errors.…”

Section: E Coli Exonucleases: Properties Structure and Functionsupporting

confidence: 77%

The DNA Exonucleases of Escherichia coli

Lovett

2011

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DNA exonucleases, enzymes that hydrolyze phosphodiester bonds in DNA from a free end, play important cellular roles in DNA repair, genetic recombination and mutation avoidance in all organisms. This article reviews the structure, biochemistry and biological functions of the 17 exonucleases currently identified in the bacterium Escherichia coli. These include the exonucleases associated with DNA polymerases I (polA), II (polB) and III (dnaQ/mutD), Exonucleases I (xonA/sbcB), III (xthA), IV, VII (xseAB), IX (xni/xgdG) and X (exoX), the RecBCD, RecJ, and RecE exonucleases, SbcCD endo/exonuclease, the DNA exonuclease activities of RNase T (rnt) and Endonuclease IV (nfo) and TatD. These enzymes are diverse in terms of substrate specificity and biochemical properties and have specialized biological roles. Most of these enzymes fall into structural families with characteristic sequence motifs, and members of many of these families can be found in all domains of life.

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Section: E Coli Exonucleases: Properties Structure and Functionsupporting

confidence: 77%

The DNA Exonucleases of Escherichia coli

Lovett

2011

EcoSal Plus

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“…The mutant frequencies Ϯ SE values were as follows: The fidelity mechanism. We have suggested that the dnaX36 mutator effect is most productively interpreted in terms of a defect in the processing of terminal mismatches (misinsertion errors) produced by the Pol III ␣-subunit (1,12,18,22,23). The current data on chromosomal DNA synthesis support this proposal.…”

Section: ϫ8supporting

confidence: 76%

“…Consistent with previous observations (1,5,18,22,23), the mutant frequencies for each of the lac alleles in control (dnaX ϩ ) strains are consistently higher for leading-strand replication than for lagging-strand replication. This persistent bias is the basis for our contention that on the E. coli chromosome, lagging-strand replication is more accurate.…”

supporting

confidence: 78%

dnaX36 Mutator of Escherichia coli : Effects of the τ Subunit of the DNA Polymerase III Holoenzyme on Chromosomal DNA Replication Fidelity

et al. 2011

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The Escherichia coli dnaX36 mutant displays a mutator effect, reflecting a fidelity function of the dnaXencoded subunit of the DNA polymerase III (Pol III) holoenzyme. We have shown that this fidelity function (i) applies to both leading-and lagging-strand synthesis, (ii) is independent of Pol IV, and (iii) is limited by Pol II.The mechanisms by which organisms achieve a high accuracy of DNA replication are of ongoing interest. Replication of the chromosome of the bacterium Escherichia coli is performed by the DNA polymerase III (Pol III) holoenzyme (HE). HE is composed of 17 subunits (10 distinct), with an overall composition (␣ε) 2 ␤ 4 2 ␥␦␦Ј (27). It contains two (␣ε) polymerase core assemblies, one for the leading strand and one for the lagging strand. The ␣ subunit is the DNA polymerase, ε is the 3Ј 3 5Ј proofreading exonuclease, and is an ε-stabilizing factor (2, 17, 34). Within HE, a central role is played by the subunit ( 2 ), which has several important functions, including connecting the two polymerases, enabling coupled leadingand lagging-strand synthesis. For the fidelity of replication, most of the focus has been on the DNA polymerase III core, notably the polymerase and the proofreading subunit (26,28,29). However, it is clear that overall chromosomal fidelity is not simply a function of polymerase fidelity but also involves activities of other HE subunits-as evidenced by mutator effects associated with defects in such subunits (24, 30, 32)-and the participation of accessory DNA polymerases (1, 8, 9, 12-14, 23, 35), of which E. coli has four (Pol I, II, IV, and V).The present study is concerned with the fidelity role of the central subunit of HE, encoded by the dnaX gene, which also encodes the ␥ subunit (6). is the full-length product of the gene (643 amino acids), while the ␥ subunit is an early termination product (residues 1 to 430). Since and ␥ share the N-terminal protein sequence (domains I, II, and III), they share certain functions, such as the loading and unloading of the ␤-processivity clamps (27). However, the two additional domains (IV and V) that are present in permit it to perform certain unique functions. Specifically, domain IV contains the site of interaction with the DnaB helicase (10), which positively regulates the speed of the replication fork (4). Domain V contains the -␣ interaction site that enables HE to be dimeric (11). Domain V also controls the cycling of the lagging-strand polymerase, as it mediates the release of the Pol III core from its ␤ processivity clamp upon completion of Okazaki fragments (20, 21). Thus, is an important control element within HE that can influence polymerase behavior, and this may extend to HE fidelity.A fidelity role for the subunit was proposed based on observations of a distinct mutator activity for certain dnaX mutants (30). In particular, dnaX36 was informative since its defect (E601K) resides in domain V and hence only affects . Presumably, in the dnaX36 mutant, the -␣ interaction is altered, leading to the mutator effect. An additional ...

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“…The combination of these activities predetermines pol I to be a key component of the E. coli replication machinery by playing an essential role during Okazaki fragment processing [21], and fulfilling functions that in eukaryotes requires the coordinated action of two enzymes, pol δ and FEN1. By means of its 5′→3′ exonuclease activity, pol I removes RNA primers that are used to initiate Okazaki fragment synthesis, while via strand displacement DNA synthesis and 3′→5′ proofreading, it accurately fills the resulting gaps in the lagging strand [22]. …”

Section: Introductionmentioning

confidence: 99%

Investigating the mechanisms of ribonucleotide excision repair in Escherichia coli

Vaisman

McDonald

Noll

et al. 2014

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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Low fidelity Escherichia coli DNA polymerase V (pol V/UmuD′2C) is best characterized for its ability to perform translesion synthesis (TLS). However, in recA730 lexA(Def) strains, the enzyme is expressed under optimal conditions allowing it to compete with the cell’s replicase for access to undamaged chromosomal DNA and leads to a substantial increase in spontaneous mutagenesis. We have recently shown that a Y11A substitution in the “steric gate” residue of UmuC reduces both base and sugar selectivity of pol V, but instead of generating an increased number of spontaneous mutations, strains expressing umuC_Y11A are poorly mutable in vivo. This phenotype is attributed to efficient RNase HII-initiated repair of the misincorporated ribonucleotides that concomitantly removes adjacent misincorporated deoxyribonucleotides. We have utilized the ability of the pol V steric gate mutant to promote incorporation of large numbers of errant ribonucleotides into the E. coli genome to investigate the fundamental mechanisms underlying ribonucleotide excision repair (RER). Here, we demonstrate that RER is normally facilitated by DNA polymerase I (pol I) via classical “nick translation”. In vitro, pol I displaces 1–3 nucleotides of the RNA/DNA hybrid and through its 5′→3′ (exo/endo) nuclease activity releases ribo- and deoxyribonucleotides from DNA. In vivo, umuC_Y11A-dependent mutagenesis changes significantly in polymerase-deficient, or proofreading-deficient polA strains, indicating a pivotal role for pol I in ribonucleotide excision repair (RER). However, there is also considerable redundancy in the RER pathway in E. coli. Pol I’s strand displacement and FLAP- exo/endonuclease activities can be facilitated by alternate enzymes, while the DNA polymerization step can be assumed by high-fidelity pol III. We conclude that RNase HII and pol I normally act to minimize the genomic instability that is generated through errant ribonucleotide incorporation, but that the “nick-translation” activities encoded by the single pol I polypeptide can be undertaken by a variety of back-up enzymes.

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Role of Escherichia coli DNA polymerase I in chromosomal DNA replication fidelity

Cited by 32 publications

References 76 publications

The DNA Exonucleases of Escherichia coli

The DNA Exonucleases of Escherichia coli

dnaX36 Mutator of Escherichia coli : Effects of the τ Subunit of the DNA Polymerase III Holoenzyme on Chromosomal DNA Replication Fidelity

Investigating the mechanisms of ribonucleotide excision repair in Escherichia coli

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