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Binding specificity of Escherichia coli single-stranded DNA binding protein for the chi subunit of DNA pol III holoenzyme and PriA helicase.

ABSTRACT: The Escherichia coli single-stranded DNA binding protein (SSB) plays a central role in DNA metabolism through its high affinity interactions with ssDNA, as well as its interactions with numerous other proteins via its unstructured C-termini. Although SSB interacts with at least 14 other proteins, it is not understood how SSB might recruit one protein over another for a particular metabolic role. To probe the specificity of these interactions, we have used isothermal titration calorimetry to examine the thermodynamics of binding of SSB to two E. coli proteins important for DNA replication, the chi subunit of DNA polymerase III holoenzyme and the PriA helicase. We find that an SSB tetramer can bind up to four molecules of either protein primarily via interactions with the last approximately 9 amino acids in the conserved SSB C-terminal tails (SSB-Ct). We observe intrinsic specificity for the binding of an isolated SSB-Ct peptide to PriA over chi due primarily to a more favorable enthalpic component. PriA and chi also bind with weaker affinity to SSB (in the absence of ssDNA) than to isolated SSB-Ct peptides, indicating an inhibitory effect of the SSB protein core. Although the binding affinity of SSB for both chi and PriA is enhanced if SSB is prebound to ssDNA, this effect is larger with PriA indicating a further enhancement of SSB specificity for PriA. These results also suggest that DNA binding proteins such as PriA, which also interact with SSB, could use this interaction to gain access to ssDNA by first interacting with the SSB C-termini.

SUBMITTER: Kozlov AG

PROVIDER: S-EPMC2861366 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Binding specificity of Escherichia coli single-stranded DNA binding protein for the chi subunit of DNA pol III holoenzyme and PriA helicase.

Kozlov Alexander G AG Jezewska Maria J MJ Bujalowski Wlodzimierz W Lohman Timothy M TM

Biochemistry 20100501 17

The Escherichia coli single-stranded DNA binding protein (SSB) plays a central role in DNA metabolism through its high affinity interactions with ssDNA, as well as its interactions with numerous other proteins via its unstructured C-termini. Although SSB interacts with at least 14 other proteins, it is not understood how SSB might recruit one protein over another for a particular metabolic role. To probe the specificity of these interactions, we have used isothermal titration calorimetry to exam ...[more]

PMID: 20329707

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Project description:Single-stranded DNA-binding protein (SSB) and PriA helicase play important roles in bacterial DNA replication restart process. The mechanism by which PriA helicase is bound and stimulated by SSB in Escherichia coli (Ec) has been established, but information on this process in Gram-positive bacteria are limited. We characterized the properties of SSB from Staphylococcus aureus (SaSsbA, a counterpart of EcSSB) and analyzed its interaction with SaPriA. The gel filtration chromatography analysis of purified SaSsbA showed a stable tetramer in solution. The crystal structure of SaSsbA determined at 1.82 Å resolution (PDB entry 5XGT) reveals that the classic oligonucleotide/oligosaccharide-binding folds are formed in the N-terminal DNA-binding domain, but the entire C-terminal domain is disordered. Unlike EcSSB, which can stimulate EcPriA via a physical interaction between EcPriA and the C-terminus of EcSSB (SSB-Ct), SaSsbA does not affect the activity of SaPriA. We also found that SaPriA can be bound by SaSsbA, but not by SaSsbA-Ct. Although no effect was found with SaSsbA, SaPriA can be significantly stimulated by the Gram-negative Klebsiella pneumoniae SSB (KpSSB). In addition, we found that the conserved SSB-Ct binding site of KpPriA (Trp82, Tyr86, Lys370, Arg697, and Gln701) is not present in SaPriA. Arg697 in KpPriA is known to play a critical role in altering the SSB35/SSB65 distribution, but this corresponding residue in SaPriA is Glu767 instead, which has an opposite charge to Arg. SaPriA E767R mutant was constructed and analyzed; however, it still cannot be stimulated by SaSsbA. Finally, we found that the conserved MDFDDDIPF motif in the Gram-negative bacterial SSB is DISDDDLPF in SaSsbA, i.e., F172 in EcSSB and F168 in KpSSB is S161 in SaSsbA, not F. When acting with SaSsbA S161F mutant, the activity of SaPriA was dramatically enhanced elevenfold. Overall, the conserved binding sites, both in EcPriA and EcSSB, are not present in SaPriA and SaSsbA, thereby no stimulation occurs. Our observations through structure-sequence comparison and mutational analyses indicate that the case of EcPriA-EcSSB is not applicable to SaPriA-SaSsbA because of inherent differences among the species.

Project description:BACKGROUND: Bacterial DNA replication restart pathways facilitate reinitiation of DNA replication following disruptive encounters of a replisome with DNA damage, thereby allowing complete and faithful duplication of the genome. In Neisseria gonorrhoeae, the primosome proteins that catalyze DNA replication restart differ from the well-studied primosome proteins of E. coli with respect to the number of proteins involved and the affinities of their physical interactions: the PriA:PriB interaction is weak in E. coli, but strong in N. gonorrhoeae, and the PriB:DNA interaction is strong in E. coli, but weak in N. gonorrhoeae. In this study, we investigated the functional consequences of this affinity reversal. RESULTS: We report that N. gonorrhoeae PriA's DNA binding and unwinding activities are similar to those of E. coli PriA, and N. gonorrhoeae PriA's helicase activity is stimulated by its cognate PriB, as it is in E. coli. This finding is significant because N. gonorrhoeae PriB's single-stranded DNA binding activity is weak relative to that of E. coli PriB, and in E. coli, PriB's single-stranded DNA binding activity is important for PriB stimulation of PriA helicase. Furthermore, a N. gonorrhoeae PriB variant defective for binding single-stranded DNA can stimulate PriA's helicase activity, suggesting that DNA binding by PriB might not be important for PriB stimulation of PriA helicase in N. gonorrhoeae. We also demonstrate that N. gonorrhoeae PriB stimulates ATP hydrolysis catalyzed by its cognate PriA. This activity of PriB has not been observed in E. coli, and could be important for PriB stimulation of PriA helicase in N. gonorrhoeae. CONCLUSIONS: The results of this study demonstrate that a bacterial PriB homolog with weak single-stranded DNA binding activity can stimulate the DNA unwinding activity of its cognate PriA helicase. While it remains unclear if N. gonorrhoeae PriB's weak DNA binding activity is required for PriB stimulation of PriA helicase, the ability of PriB to stimulate PriA-catalyzed ATP hydrolysis could play an important role. Thus, the weak interaction between N. gonorrhoeae PriB and DNA might be compensated for by the strong interaction between PriB and PriA, which could result in allosteric activation of PriA's ATPase activity.

Dataset Information

Binding specificity of Escherichia coli single-stranded DNA binding protein for the chi subunit of DNA pol III holoenzyme and PriA helicase.

Publications

Binding specificity of Escherichia coli single-stranded DNA binding protein for the chi subunit of DNA pol III holoenzyme and PriA helicase.

Similar Datasets

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