Escherichia coli DNA polymerase II is stimulated by DNA polymerase III holoenzyme auxiliary subunits

Hughes, Ami; Bryan, Sharon K.; Chen, Hong; Moses, Robb E.; McHenry, Charles S.

doi:10.1016/s0021-9258(20)64360-5

Cited by 55 publications

(8 citation statements)

References 49 publications

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“…The γ complex and the β subunit of pol III were also shown to endow pol II with high processivity ( , ). Do these proteins stimulate translesion replication by pol II?…”

Section: Resultsmentioning

confidence: 99%

Analysis of Unassisted Translesion Replication by the DNA Polymerase III Holoenzyme

Tomer

Livneh²

1999

Biochemistry

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DNA damage-induced mutations are formed when damaged nucleotides present in single-stranded DNA are replicated. We have developed a new method for the preparation of gapped plasmids containing site-specific damaged nucleotides, as model DNA substrates for translesion replication. Using these substrates, we show that the DNA polymerase III holoenzyme from Escherichia coli can bypass a synthetic abasic site analogue with high efficiency (30% bypass in 16 min), unassisted by other proteins. The theta and tau subunits of the polymerase were not essential for bypass. No bypass was observed when the enzyme was assayed on a synthetic 60-mer oligonucleotide carrying the same lesion, and bypass on a linear gapped plasmid was 3-4-fold slower than on a circular gapped plasmid. There was no difference in the bypass when standing-start and running-start replication were compared. A comparison of translesion replication by DNA polymerase I, DNA polymerase II, the DNA polymerase III core, and the DNA polymerase III holoenzyme clearly showed that the DNA polymerase III holoenzyme was by far the most effective in performing translesion replication. This was not only due to the high processivity of the pol III holoenzyme, because increasing the processivity of pol II by adding the gamma complex and beta subunit, did not increase bypass. These results support the model that SOS regulation was imposed on a fundamentally constitutive translesion replication reaction to achieve tight control of mutagenesis.

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“…The γ complex and the β subunit of pol III were also shown to endow pol II with high processivity ( , ). Do these proteins stimulate translesion replication by pol II?…”

Section: Resultsmentioning

confidence: 99%

Analysis of Unassisted Translesion Replication by the DNA Polymerase III Holoenzyme

Tomer

Livneh²

1999

Biochemistry

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“…The processivity proteins of E. coli, the β subunit sliding DNA clamp and the γ complex, are essential for replication by pol III, endowing it with remarkable processivity (25). Moreover, in vitro experiments have shown that these proteins increase the processivity of other polymerases, including pol II (26,27), pol IV (7,28), and pol V (7; this study), although the in vivo significance of these effects is not yet known. As far as pol V is concerned, it was reported that lesion bypass by UmuD′ 2 C, RecA, and SSB was negligible, unless the β subunit and the γ complex were added (5,7).…”

Section: Discussionmentioning

confidence: 90%

“…Although they were originally discovered as the processivity subunits of pol III, it is clear now that the β subunit and the γ complex increase the processivity of pol II (26,27), pol IV (7,28), and pol V (7; this study). There is also evidence for an interaction between the β clamp and pol I, and increased activity of pol I was observed in the presence of the processivity subunits, which is likely explained by an increase in the processivity of pol I (29).…”

Section: Acknowledgmentmentioning

confidence: 87%

Analysis of the Stimulation of DNA Polymerase V of Escherichia coli by Processivity Proteins

Maor-Shoshani

Livneh

2002

Biochemistry

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Bypass of replication-blocking lesions in Escherichia coli is carried out by DNA polymerase V (UmuC) in a reaction that requires UmuD', RecA, and single-strand DNA-binding protein (SSB). The activity of this four-component basic bypass system is a low-fidelity and low-processivity activity. Addition of the processivity subunits of pol III, the beta subunit sliding DNA clamp, and the five-subunit gamma complex clamp loader increased the rate of translesion replication approximately 3-fold. This stimulation was specific to the lesion bypass step, with no effect on the initiation of synthesis by pol V. The beta subunit and gamma complex increased the processivity of pol V from 3 to approximately 14-18 nucleotides, providing a mechanistic basis for their stimulatory effect. Stimulation of bypass was observed over a range of RecA and SSB concentrations. ATPgammaS, which strongly inhibits translesion replication by pol V, primarily via inhibition of the initiation stage, caused the same inhibition also in the presence of the processivity proteins. The in vivo role of the processivity proteins in translesion replication was examined by assaying UV mutagenesis. This was done in a strain carrying the dnaN59 allele, encoding a temperature-sensitive beta subunit. When assayed in an excision repair-defective background, the dnaN59 mutant exhibited a level of UV mutagenesis reduced up to 3-fold compared to that of the isogenic dnaN(+) strain. This suggests that like in the in vitro system, the beta subunit stimulates lesion bypass in vivo.

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“…Clamp proteins slide on DNA and serve as platforms for multiple proteins to bind. The b-clamp interacts with all five E. coli DNA polymerases (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) and coordinates the polymerase switch during translesion synthesis (27)(28)(29). In addition, b interacts with replication initiation factors DnaA (30) and Had (31); mismatch repair proteins MutS and MutL; and DNA ligase A, which is responsible for Okazaki fragment maturation (16).…”

Section: Introductionmentioning

confidence: 99%

Dynamics of the E. coli β-Clamp Dimer Interface and Its Influence on DNA Loading

Koleva

Gökcan

Rizzo

et al. 2019

Biophysical Journal

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The ring-shaped sliding clamp proteins have crucial roles in the regulation of DNA replication, recombination, and repair in all organisms. We previously showed that the Escherichia coli b-clamp is dynamic in solution, transiently visiting conformational states in which Domain 1 at the dimer interface is more flexible and prone to unfolding. This work aims to understand how the stability of the dimer interface influences clamp-opening dynamics and clamp loading by designing and characterizing stabilizing and destabilizing mutations in the clamp. The variants with stabilizing mutations conferred similar or increased thermostability and had similar quaternary structure as compared to the wild type. These variants stimulated the ATPase function of the clamp loader, complemented cell growth of a temperature-sensitive strain, and were successfully loaded onto a DNA substrate. The L82D and L82E I272A variants with purported destabilizing mutations had decreased thermostability, did not complement the growth of a temperature-sensitive strain, and had weakened dimerization as determined by native trapped ion mobility spectrometry-mass spectrometry. The b L82E variant had a reduced melting temperature but dimerized and complemented growth of a temperature-sensitive strain. All three clamps with destabilizing mutations had perturbed loading on DNA. Molecular dynamics simulations indicate altered hydrogen-bonding patterns at the dimer interface, and cross-correlation analysis showed the largest perturbations in the destabilized variants, consistent with the observed change in the conformations and functions of these clamps.

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Escherichia coli DNA polymerase II is stimulated by DNA polymerase III holoenzyme auxiliary subunits

Cited by 55 publications

References 49 publications

Analysis of Unassisted Translesion Replication by the DNA Polymerase III Holoenzyme

Analysis of Unassisted Translesion Replication by the DNA Polymerase III Holoenzyme

Analysis of the Stimulation of DNA Polymerase V of Escherichia coli by Processivity Proteins

Dynamics of the E. coli β-Clamp Dimer Interface and Its Influence on DNA Loading

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