Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Involvement of histidine in complex formation of PriB and single-stranded DNA
Graphical abstract
Introduction
In Escherichia coli, DNA replication fork, starting at the replication origin (oriC), is occasionally arrested upon encountering chemically damaged DNA [1] and the damage is repaired by DNA repair proteins through various mechanisms. After DNA is repaired, its replication restarts. During DNA replication, initiation is strictly regulated by a specific DNA sequence in oriC [2]; however, after DNA repair, replication restart is a DNA-sequence-independent system, where several replication restart proteins assemble. This system is called the replication restart primosome [3], [4]. Restart systems that depend on this primosome are mainly divided into two alternative systems, namely, 1) the PriA-dependent system and 2) the PriC-dependent system [3], [5], [6], [7]. In the PriA-dependent system, the N-terminal domain of PriA recognized the 3′-end of the nascent leading strand DNA in the repaired fork and PriA stabilized the repaired fork structure [8], [9]. The unwound single-stranded DNA (ssDNA) in the lagging strand provides a scaffold of DnaC and DnaB helicase loading [6], [10]. DnaG primase then synthesizes the primer RNA. Finally, from the primed sites, DNA chains are extended by a DNA polymerase III holoenzyme. These processes are a series of restart replications of genomic DNA.
PriB, a basic 10-kDa protein, is considered an accessory protein during PriA-dependent restart [3], [11]. It has a role in stabilizing the PriA–DNA complex [12], [13] and increasing PriA helicase activity [14], [15]. PriB also interacts with DnaT, which may recruit the DnaB–DnaC complex [10], [16], [17]. Therefore, PriB has an important role in the replication restart primosome. The X-ray structure of PriB monomer represents an OB (oligonucleotide/oligosaccharide binding) fold structure such as SSB (single-stranded DNA binding protein) [18], [19], [20]. Gene analysis also suggests this similarity, i.e., that PriB is derived from the duplication of the SSB gene [21]. In contrast to these similarities, SSB is a tetramer unit [22], [23] while PriB represents a symmetric dimer that is conserved in several eubacteria [24], [25]. Both PriB and SSB possess ssDNA binding ability [18], [19], [20], [26]. Although the SSB tetramer binds to ssDNA as a unit, the PriB–DNA complex structure of the 15-mer oligo-dT and PriB reveals the possibility that two ssDNAs bind to the PriB dimer [27]. Moreover, the PriB dimer binds to ssDNA through their grooves, mainly electropositive residues in β4–loop3–β5 because there is loss of function with amino acid substitutions that maintain these electropositive interactions [10], [27]. Therefore, the structure of the PriB monomer is similar to that of SSB; however, the manners of DNA binding and oligomerization between PriB and SSB differ.
SSB has ssDNA binding ability with positive cooperativity [28], [29], [30]. In E. coli, SSB facilitates ‘unlimited cooperative binding’ and ‘limited cooperative binding’, depending on the salt concentration. On the other hand, PriB–ssDNA interaction also reveals positive cooperativity dependent on ssDNA length and salt concentration [27], [31]. A highly cooperative binding of PriB to homopolymers longer than 24–30 bases occurs. These reports suggest that positive cooperativity arises in the sandwiched DNA structure by two PriB dimer molecules, as observed in the asymmetric unit of the X-ray crystal structure. However, the cooperative mechanism underlying ssDNA binding to PriB has not been elucidated, as biochemical studies have yet to provide sufficient evidence.
In the present study, we examined the PriB and 35-mer ssDNA interactions using NMR. The results suggested two binding modes for ssDNA binding to PriB. In the primary binding mode, single-stranded DNA bound to the electropositive groove of PriB. Next, the secondary binding mainly occurred around the α-helix region in PriB. FRET assays indicated that positive charges of histidine residues in low pH contributed to the second binding and the compact formation by PriB interaction. Furthermore, amino acid substitution of His residues in PriB suggested that His64 in the α-helix region was involved in PriB–ssDNA interaction. Thus, based on the structure, we proposed the role of His64 in PriB–ssDNA interaction and the relationship between second binding and cooperativity in PriB molecules.
Section snippets
Protein preparation of PriB
The expression and purification of PriB were performed according to a previous method with slight modification [20]. Although we used spermidine for removal of DNA, it tends to increase insoluble aggregation; therefore, to increase the recovery yields of PriB, we did not use spermidine in our purification. When we treated PriB with 1 M NaCl instead of spermidine, we yielded 20 mg/L PriB in LB culture. For the preparation of 15N- or/and 13C-labeled protein, pET22b-PriB-transformed cells were
Main-chain assignment of PriB
To examine the interaction between ssDNA with PriB, we prepared the 13C, 15N double-labeled PriB protein. The 1H–15N HSQC spectrum of purified 13C, 15N double-labeled PriB in aqueous solution was recorded at 25 °C and pH 6.5 containing 200 mM (NH4)2SO4 (Supplementary Fig. 1). The spectrum consisted of separated and dispersed peaks. Using the double-labeled sample and heteronuclear 3D NMR, we assigned the 1H, 15N, and 13C chemical shifts of the main-chain of PriB with the exceptions of Thr2, Asn3,
Discussion
In the present study, we assigned chemical shifts of PriB protein and examined the ssDNA interaction site in the PriB structure using chemical shift perturbation. The chemical shift perturbations that occurred with the addition of oligo-dT7 and -dT15 were observed as almost linear, whereas the chemical shift changes in the primary and secondary modes occurred by the addition of oligo-dT35. Moreover, FRET measurements of oligo-dT35 modified with 5′-Cy3 and 3′-Cy5 represented a two-step
Acknowledgements
This work was supported by KAKENHI (23570140) to Y.A. and T.K. and by a grant-in-aid for JSPS Fellows to A. T. We thank Prof. Daisuke Kohda of Kyushu University for the fluorescence anisotropy experiment and Dr. Hiroaki Yokota of Kyoto University for his suggestions about the FRET experiment. We also thank KN International and Editage service by Cactus Communications Inc. for improving the English usage in our manuscript.
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2019, Journal of Biological ChemistryCitation Excerpt :The DnaT–PriA–DNA complex could then utilize the ssDNA site to facilitate replicative helicase (DnaB in E. coli) loading. Considering that the ssDNA site size of PriA on the gapped DNA used in this study is 5 nucleotides and the ssDNA site sizes of PriB, DnaT, and DnaB are 8–12 (62, 63), ∼25 (61), and ∼20 nucleotides (65), respectively, PriA helicase activity and PriB may function together to create and protect the ssDNA for DnaT binding (and ultimately DnaB binding). Additionally, the β-hairpin and PriB could function in displacement of proteins, such as SSB, from the template-lagging strand (Fig. 8B).
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These authors contributed equally to this work.