Substitutions of Conserved Residues in the C-terminal Region of DnaC Cause Thermolability in Helicase Loading

Magdalena M Felczak,Jay M Sage,Katarzyna Hupert-Kocurek,Senem Aykul,Jon M Kaguni

doi:10.1074/jbc.m115.708586

Magdalena M Felczak, Jay M Sage + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m115.708586

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Abstract

The DnaB-DnaC complex binds to the unwound DNA within the Escherichia coli replication origin in the helicase loading process, but the biochemical events that lead to its stable binding are uncertain. This study characterizes the function of specific C-terminal residues of DnaC. Genetic and biochemical characterization of proteins bearing F231S and W233L substitutions of DnaC reveals that their activity is thermolabile. Because the mutants remain able to form a complex with DnaB at 30 and 37 °C, their thermolability is not explained by an impaired interaction with DnaB. Photo-cross-linking experiments and biosensor analysis show an altered affinity of these mutants compared with wild type DnaC for single-stranded DNA, suggesting that the substitutions affect DNA binding. Despite this difference, their activity in DNA binding is not thermolabile. The substitutions also drastically reduce the affinity of DnaC for ATP as measured by the binding of a fluorescent ATP analogue (MANT-ATP) and by UV cross-linking of radiolabeled ATP. Experiments show that an elevated temperature substantially inhibits both mutants in their ability to load the DnaB-DnaC complex at a DnaA box. Because a decreased ATP concentration exacerbates their thermolabile behavior, we suggest that the F231S and W233L substitutions are thermolabile in ATP binding, which correlates with defective helicase loading at an elevated temperature.

Highlights

DNA replication in Escherichia coli starts at the chromosomal replication origin through a series of discrete biochemical events
The F231S and W233L Substitutions Confer Thermolability in DNA Replication at 37 °C—Using a genetic assay that measures the function of plasmid-borne dnaC alleles to complement a null dnaC strain at 37 °C [17], we identified novel dnaC mutations (Fig. 1)
We focused on mutations encoding F231S and W233L substitutions and found that both were defective in this genetic assay at 37 °C but remained active at 30 °C (Table 1)

Summary

Experimental Procedures

Reagents and Proteins—Replication proteins have been described [13, 20]. Mutant DnaC proteins were purified essentially as described after their induced expression [13], but the host strain was E. coli Lemo (DE3). Isolation and Genetic Analysis of dnaC Alleles—The genetic method to identify defective dnaC mutations relies on E. coli MF1061 (araD139 ⌬(ara, leu)7697 ⌬lacX74 galU galK rpsL hsdR2 (rKϪ mKϩ) mcrB1 ⌬dnaC::cat recA635::kan) carrying the plasmid pAM34dnaCL1– 4 that encodes the dnaCϩ gene [17] This plasmid, which confers ampicillin resistance and complements the ⌬dnaC::cat mutation of the host strain, requires IPTG in the culture medium for its maintenance. To measure DNA replication, the indicated void volume fractions (20 ␮l) were supplemented with single-stranded DNA-binding protein, primase, DNA polymerase III holoenzyme, magnesium acetate (10 mM final concentration), and other required components at standard amounts for M13 A site ssDNA replication in a final volume of 25 ␮l, followed by incubation at 30 °C or 37 °C for 10 min, which showed that assembly of the ABC complex is thermolabile..

Results

Relative plating efficiencya

Response Units

Supercoiled Form I*

Discussion