Characterization of Streptococcus pneumoniae PriA helicase and its ATPase and unwinding activities in DNA replication restart.

DNA replication forks often encounter template DNA lesions that can stall their progression. The PriA-dependent pathway is the major replication restart mechanism in Gram-positive bacteria, and it requires several primosome proteins. Among them, PriA protein-a 3' to 5' superfamily-2 DNA helicase-is the key factor in recognizing DNA lesions and it also recruits other proteins. Here, we investigated the ATPase and helicase activities of Streptococcus pneumoniae PriA (SpPriA) through biochemical and kinetic analyses. By comparing various DNA substrates, we observed that SpPriA is unable to unwind duplex DNA with high GC content. We constructed a deletion mutant protein (SpPriAdeloop) from which the loop area of the DNA-binding domain of PriA had been removed. Functional assays on SpPriAdeloop revealed that the loop area is important in endowing DNA-binding properties on the helicase. We also show that presence of DnaD loader protein is important for enhancing SpPriA ATPase and DNA unwinding activities.


Introduction
Successful chromosomal DNA replication is a crucial process for cell growth and proliferation. However, DNA replication forks often encounter lesions in the DNA template, arising from diverse conditions like ultraviolet irradiation or other damaging factors, which can stall replication progression [1]. Stalled replication can result in incomplete chromosome duplication, genomic instability, and cell death in both bacteria and eukaryotes [2][3][4][5][6]. Therefore, stalled replication forks must be efficiently repaired to allow resumption of genome replication.
In bacteria, DNA replication restart is regulated by a group of proteins called primosomal proteins [7][8][9]. In Gram-negative bacteria (such as Escherchia coli), PriA protein recognizes stalled forks and sequentially recruits other primosome members (PriB, PriC and DnaT) and a loader protein (DnaC) to assist the replicative hexameric helicase DnaB to restart the replication process [10][11][12][13][14][15][16]. However, the essential components PriB, PriC and DnaT are not found in Gram-positive bacteria. Instead, DNA replication restart in Gram-positive bacteria (such as Bacillus subtilis) is controlled by assembly of primosome proteins PriA, DnaD, DnaB, and the DnaC/I complex (homologous to the DnaB/C complex of Gram-negative bacteria) into a functional primosome through a series of ordered protein-protein interactions at a repaired DNA replication fork site [17,18,8]. Since the primosome members of Gram-negative and Gram-positive bacteria seem to differ prior to recruitment of the replicative helicase, their mechanisms of primosome assembly and function appear to have diverged somewhat [15,19,20]. Although PriA mediates the primosome assembly processes of both Gram-negative and Gram-positive bacteria, most studies of the PriA-mediated DNA replication restart pathway have focused on well-studied Gram-negative bacteria. Therefore, it is worth investigating this topic in Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200269/894011/bcj-2020-0269.pdf by guest on 29 September 2020 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BCJ20200269 Gram-positive bacteria.
PriA is a 3′-to-5′ DNA helicase of the SF2 helicase superfamily and plays two major roles in bacterial DNA replication restart [21]; the first is binding and remodeling of stalled forked structures to generate a single-stranded region on the lagging-strand template for replicative hexameric helicase binding, and the second is to orchestrate origin-independent replisome loading at the stalled fork [19]. PriA helicases comprise five well-conserved helicase motifs; Walker A, Walker B, SAT motif, Cys-rich region and motif VI [22]. These motifs facilitate its DNA remodeling activity at a repaired DNA site by unwinding short fragments of paired DNA for origin-independent assembly of a new replisome at the stalled fork [21,23,24].
Furthermore, recent 3D structural study of Gram-negative Klebsiella pneumoniae PriA helicase (KpPriA) has shown that PriA has several clustered subdomains: a 3′ DNA-binding domain (3′ BD), an unusual circularly-permuted winged-helix domain at the N-terminal DNA binding domain (DBD), and a helicase domain (HD) consisting of a Cys-rich region and a C-terminal domain [25]. Although PriA is well-conserved among sequenced bacterial genomes [23], the composition of primosome members and the functions of the individual primosome proteins differ among species. Thus, establishing the precise differences in PriA protein among species is of considerable interest.
Here, we studied the PriA protein from an important Gram-positive bacterium, Streptococcus pneumoniae. S. pneumoniae is a pathogen and its antibiotic-resistance is becoming more prevalent worldwide [26], so new treatments for S. pneumoniae infection are required. Successful replication is thought to be important for the pathogenicity of certain bacteria such as S. pneumoniae [26]. In addition, PriA has been proven to play a critical role in DNA repair and resistance to damaging factors Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200269/894011/bcj-2020-0269.pdf by guest on 29 September 2020 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BCJ20200269 such as UV irradiation or high temperatures [6]. We sought to examine the mechanisms underlying S. pneumoniae PriA (SpPriA) activity by applying several biochemical assays. Our results revealed that SpPriA has some surprising DNA unwinding abilities.

Plasmid construction
The artificial genes encoding S. pneumoniae priA (priA and priA deloop ), ssbA and dnaD genes (dnaD, dnaD-Ntd and dnaD-Ctd) that were codon-optimized for expression in E. coli were purchased from an external gene synthesis service (Protech Technology Enterprise Co. Ltd., Taipei, Taiwan). The priA gene of E. coli was amplified from chromosomal DNA by PCR. These genes were cloned into pET21b (Novagen) vector with a C-terminal His 6 tag for protein expression in E. coli BL21 (DE3). To remove the His 6 -tag, we cloned the priA gene of S. pneumoniae into pSol-His vector (Lucigen) with a TEV cutting site and transformed it into E. coli BL21 (DE3) for protein expression.

Protein expression and purification
The SpPriA, SpPriA deletion mutant (SpPriA deloop ), and EcPriA plasmids were transformed and overexpressed in E. coli BL21 (DE3). Transformed E. coli were cultured in LB medium containing 100 µg/ml ampicillin and grown at 37 °C. Once the optical density (at 600 nm) of the culture reached 0.8, 1 mM IPTG was added. The culture of SpPriA was then incubated at 20 °C for 12 h. Cells were harvested by centrifugation at 4500 g for 30 min at 4 °C for the following purification steps.
Cell pellets were suspended in buffer A containing 20 mM Tris-HCl pH8.0, 500

ATPase assay
Steady-state ATPase activity assays were performed according to the malachite green method [27], but with slight modifications. We incubated 50 nM PriA with 500 nM DNA substrate and ATP (at indicated concentrations) in 20 mM Tris-HCl pH8.0, 50 mM NaCl, 5 mM MgCl 2, and 2 mM DTT. The assay was carried out in a final volume of 200 μl of SpPriA-DNA reaction mixture for 30 min at 37 °C. We added 200 μl 10% SDS to terminate the reaction, and then used 1.25% ammonium molybdate in 6.5% H 2 SO 4 (200 μl) and 9% ascorbic acid (200 μl) to detect the phosphate product.
Released free phosphate and molybdic acid form a complex that can be reduced to generate a deep blue color recorded at 660 nm. The assays were corrected for spontaneous ATP degradation. Under our standard assay conditions, ATPase activity is defined as pmol ATP pmol PriA -1 min -1 . The impact of 200 nM SpDnaD and its Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200269/894011/bcj-2020-0269.pdf by guest on 29 September 2020 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BCJ20200269 truncated proteins on the ATPase activity of SpPriA (50 nM) was also investigated.
The kinetic parameters K m and k cat were determined with respect to ATP by fitting the ATPase hydrolysis rates to the Michaelis-Menten equation, where S = ATP. Values for k cat were determined by dividing V max by the concentration of SpPriA in the reaction. Three independent repeats for SpPriA ATPase activity were conducted and associated uncertainties are represented as standard deviations of the mean.

Helicase activity assays
DNA substrates were prepared by annealing a 5′ Cyanine-3-labeled DNA with its complementary DNA to generate a duplex DNA with a 3′ overhang or a Y-shaped DNA (Supplementary Table S1). The annealed double-stranded DNA (dsDNA) substrates were further purified through polyacrylamide gels by the crush and soak method with slight modification [28]. Briefly, the excised gel slice was crushed into tiny pieces and placed in a tube. Sufficient buffer was added to the gel pieces to cover them, and this slurry was then incubated with shaking for 16 h at room temperature to achieve better recovery. The slurry was then centrifuged and the supernatant was Unlabeled DNA (200 nM) was added to the reaction to anneal to unwound DNA generated by PriA helicase activity. We added 100 µg/ml Proteinase K and incubated Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200269/894011/bcj-2020-0269.pdf by guest on 29 September 2020 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BCJ20200269 the reaction solution for 30 min at 37 °C. Native gel running dye (10 mM Tris-HCl pH7.6 and 5% glycerol final concentration) was directly added to samples before loading onto 10% TBE polyacrylamide gels for electrophoresis in 0.5X TBE buffer (45 mM Tris-borate, 1 mM ethylene diamine tetraacetic acid (EDTA), pH8.0) for 3 h at 70 V. Gels were immediately scanned for fluorescence signals using the Cy3 channel with a Typhoon FLA9000 system (GE Healthcare) to visualize DNA bands.

SpPriA ATPase activity
To characterize the function of SpPriA, we performed PriA ATPase activity assays.
Since the ATPase activity of superfamily 2 helicases is known to be strongly dependent on the presence of DNA [29,30], we used various DNA molecules, including short single-stranded oligonucleotides, linear 3′ overhang dsDNA, and Y-forked dsDNA (Supplementary Table S1). In addition, the linear 3′ overhang dsDNA and Y-forked dsDNA have different base-pairing sequences and different free single-stranded tail lengths (10-nucleotides in E20L, 30-nucleotides in Y30H). As shown in Figure 1, each of these DNA substrate types could stimulate ATPase activity by SpPriA, whereas negligible ATPase activity was observed in the absence of DNA.
Moreover, our results show that SpPriA ATPase activity was greater for longer single-stranded DNA (ssDNA), and that the ATPase activity of Y-forked dsDNA was greater than that of 3′ overhang dsDNA. Therefore, the size and structural features of DNA have an impact on SpPriA ATPase activity. We also performed a kinetic activity assay for SpPriA in the presence of Y30H (a Y-forked dsDNA), but in the absence of the accessory loader protein SpDnaD. The kinetic parameters K m and k cat for SpPriA were 0.44 mM and 14.65 M -1 s -1 , respectively (Table 1). Next, we compared the

SpPriA exhibits only slight helicase activity on DNA with high GC content
Since the major function of PriA is to act as a 3′-to-5′ helicase [22], we examined the DNA unwinding ability of SpPriA. Surprisingly, our His-tagged purified SpPriA showed no unwinding activity on a variety of DNA substrates (shown in the left panels of Figure 2). The His 6 -tag may have altered SpPriA enzymatic activity, resulting in the lack of helicase activity for several tested dsDNA substrates. To elucidate if the His-tag affects SpPriA activity, we removed the His 6 -tag from SpPriA His-tag retains complete activity in vivo remains to be tested.
A previous study revealed that PriA from Deinococcus radiodurans (DrPriA) is a pseudohelicase due to a mutation from lysine to arginine of a critical catalytic amino acid residue in the Walker A motif (also known as the phosphate-binding loop or P-loop) [31]. This amino acid substitution has been introduced into E. coli (the K230R mutant of EcPriA), and the resulting mutant helicase cannot hydrolyze ATP or unwind DNA substrate [22]. However, we found that this important lysine residue is conserved in SpPriA (Supplementary Figure S1).
To determine why SpPriA shows no unwinding activity in the presence of certain DNA substrates and when ATP and magnesium ions are also present, we examined other PriA helicase motifs that are also important for unwinding duplex DNA. Apart from the Walker A motif, there are another four well-conserved helicase motifs among sequenced bacterial PriA homologs: Walker B, SAT motif, Cys-rich motif, and motif VI [22]. The Walker B motif is involved in binding an active Mg 2+ and directs interaction with DNA. The SAT and Cys-rich motifs are involved in helicase activity but not ATPase activity. Motif VI (QxxGRxGR) is associated with both ATPase and helicase activities [32,33]. However, our sequence alignment (Supplementary Figure   S1) revealed broad sequence conservation in these helicase motifs between SpPriA and other homologs.
Since SpPriA showed ATPase activity but lacked apparent unwinding activity on forked or 3' overhang dsDNA, we speculated that the size, GC content or structural features of the DNA substrate may curtail SpPriA unwinding activity. Therefore, we further tested SpPriA unwinding activity on additional DNA substrates (see Supplementary Table S1) and found that it could only unwind DNA substrates having low GC content such as E20L, Y30L, E60L and Y60L (right panels of Figure 2). Interestingly, the major difference between the substrates shown in the left and right panels of Figure 2 is their GC contents (~45% for E20L, Y30L, E60L and Y60L and ~77% for E20H, Y30H, E60H and Y60H). Both non-ring and hexameric helicases exhibit moderate to high sensitivity to DNA substrates with high GC contents [34,29,[35][36][37]. Since helicase motifs are highly conserved among PriA homologs, it raises the intriguing question as to why SpPriA can only unwind DNA substrates with low GC content. GC contents of bacterial genomes are dramatically diverse, ranging from less than 20% to more than 70%, but the reasons for this variation remain unclear. A previous report showed that the GC content of the S. pneumoniae genome is low (39.7%) [38]. Potentially, the low GC content of the S. pneumoniae genome may explain why SpPriA is less efficient at unwinding DNA substrates with high GC contents.

DNA-binding ability of SpPriA
To establish if the lack of unwinding activity by SpPriA is due to deficient DNA-binding ability, we measured its DNA-binding affinity using various 5′-end Cy3-labeled DNA substrates by means of a fluorescence polarization-based equilibrium DNA-binding assay. The DNA substrates we assessed included ssDNAs, partial duplex DNAs with a 3′ ssDNA overhang, and forked dsDNA structures (Supplementary Table S1). These DNA substrates were incubated with increasing concentrations of SpPriA proteins and their fluorescence polarization signals were recorded to evaluate DNA-protein interactions. The binding curves and dissociation constants (Kd) are shown in Figure 3. We found SpPriA binding to each of the DNA substrates to be concentration-dependent, which is consistent with previous studies of exhibited the highest binding affinities for the Y-forked dsDNA substrates, with binding constants for the partial duplex DNAs with a 3′ overhang being higher than for ssDNA substrates (Figure 3). Moreover, binding constants were higher for the long ssDNA fragments (dT50) than for the shorter fragments (dT20) (Figure 3). Surprisingly, SpPriA binding affinities did not appear to be correlated with GC contents. Together, these data indicate that SpPriA's inability to unwind some DNA substrates is not due to defective DNA binding.
Our ATPase assays had shown that size and structural elements of DNA impact PriA ATPase activity (Figure 1), yet there were no significant differences between DNA substrates harboring high or low GC content (Supplementary Figure S6). Therefore, the lower efficiency of SpPriA for unwinding DNA substrates with high GC content is not due to a lack of or inefficient DNA-binding or ATPase activity, instead suggesting that other elements control GC content sensitivity. interactions between these two structural elements being important for DNA substrate recognition and binding [44,45]. Notably, the length of this connecting loop is much shorter (by ~30 residues) in Gram-negative bacteria than in Gram-positive bacteria.

Characterization of a truncated
Overall mean sequence similarity among our aligned PriA sequences is 65.5%, with mean sequence similarity specifically of the PriA helicase domain being 71.6%.
However, mean sequence similarity of the WH motif is ~44% (residues 115-177 in EcPriA or 150-242 in SpPriA). These divergences between Gram-positive and Gram-negative sequences might reflect different DNA substrate recognition or binding abilities. Moreover, in Gram-positive bacteria, DnaD and DnaB proteins stimulate helicase activity by directly interacting with PriA [46,47], whereas in Gram-negative bacteria, a PriA-PriB-DNA ternary complex is formed to induce PriA helicase activity [11]. Accordingly, we constructed recombinant SpPriA deloop protein in which the connecting loop (residues 89-118) had been removed. To characterize this truncated protein, we first tested its DNA binding abilities through fluorescence-polarization DNA binding assay. We found that the DNA binding affinities of SpPriA deloop for all tested DNA substrates was significantly decreased relative to SpPriA (compare Supplementary Figure S7 to Figure 3). An  (Table 1). These data indicate that the connecting loop between the 3′ BD and WH motifs is important for DNA binding affinities.

SpDnaD significantly enhances the ATPase activity of SpPriA
Our experimental results showed that SpPriA has ATPase activity but no unwinding activity for DNA substrates with high GC content. In fact, most non-hexameric superfamily 2 DNA helicases have exhibited relatively poor helicase activity in vitro [49]. This phenomenon is not surprising because several accessory proteins significantly enhance helicase enzymatic activity through protein-protein interactions [49]. For instance, the ATPase activity of EcPriA is enhanced strongly by PriB and SSB [25,11,50]. Thus, we overexpressed and purified SpSsbA [51] and tested SpPriA ATPase and unwinding assays in the presence or absence of SpSsbA (Supplementary Figure S8). The results reveled that presence of SpSsbA neither enhanced SpPriA's ATPase activity nor its ability to unwind dsDNA with high GC content. Since DnaD and DnaB proteins are defined as co-loader proteins for the DnaC/I complex [52,18,53,54], we speculate that these two proteins play other roles in the primosome assembly process. Our previous study showed that DnaB and PriA have a direct physical interaction [47], so we wondered if SpDnaD might also enhance the enzymatic activity of SpPriA. To examine this possibility, we tested ATP hydrolysis as catalyzed by 50 nM SpPriA in the presence of 500 nM Y-forked dsDNA (Y30H) and 200 nM SpDnaD. As shown in Figure 5A, addition of SpDnaD greatly stimulated (2.1-fold enhancement effect, maximum 87.4 M ATP/min) the ATPase activity of SpPriA. Moreover, kinetic parameters showed that addition of SpDnaD had no significant effect on the K m value of SpPriA, but did strongly impact the value of k cat (Table 1). In the presence of SpDnaD, k cat of SpPriA increased to 31.14 ±0.89 from outcome (Supplementary Figure S9).
In E. coli, the ATPase and helicase activities of PriA can be stimulated through the ssDNA-binding activity of PriB [11]. To elucidate the DNA binding ability of SpDnaD, we measured SpDnaD Kd values with both ssDNA (dT30) and 3′ overhang dsDNA (E20L). As shown in Supplemental Figure  DnaD has an N-terminal domain that mediates scaffold formation [55,56] and a C-terminal domain that binds to DNA [56]. Previous studies showed that the C-terminal domain of DnaD is correlated with its DNA binding abilities [56], so based on DnaD sequence alignment and previous study [46] we generated two deletion constructs (SpDnaD-Ntd and SpDnaD-Ctd) and used fluorescence polarization to test their functional and physical interactions with SpPriA. As shown in Figure 6A, the Kd values for full-length SpDnaD and SpDnaD-Ntd with SpPriA were comparable (14.4 nM for SpDnaD FL and 103 nM for SpDnaD-Ntd). However, SpDnaD-Ctd barely bound to SpPriA (Kd is not detectable). Together, these data demonstrate that the N-terminal region of SpDnaD is required for physical interaction with SpPriA.

SpPriA requires SpDnaD to efficiently unwind duplex DNA
Several studies have shown that certain accessory proteins facilitate effective destabilization of base-paired DNA to facilitate helicase activity [34,57,35,36]. Since SpDnaD has DNA-binding affinities (Supplemental Figure S10), interacts strongly with SpPriA ( Figure 6A), and enhances SpPriA ATPase activity (Table 1), we tested if Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200269/894011/bcj-2020-0269.pdf by guest on 29 September 2020 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BCJ20200269 SpDnaD contributes to the unwinding activity of SpPriA. We assessed SpPriA helicase activity on a Y-forked dsDNA substrate (Y30H) that cannot be unwound by SpPriA in the absence of SpDnaD ( Figure 2). As shown in Figure 6B, increasing the ratio of SpDnaD to SpPriA significantly enhanced helicase activities on dsDNA in a concentration-dependent manner. Thus, SpDnaD plays a dual role, increasing both the ATPase and helicase activities of SpPriA. For this latter effect, SpDnaD may destabilize DNA base-pairing or act like SSB protein in E. coli to bind displaced ssDNA and prevent reannealing of intermediates from the unwinding reaction [50].
In summary, we have characterized the PriA protein from an important Gram-positive pathogenic bacterium, S. pneumoniae. We have shown that SpPriA is more efficient at unwinding DNA substrates with low GC contents ( Figure 2). In contrast to its activity on DNA with low GC content, SpPriA showed negligible unwinding ability on DNA with high GC content (Figure 2), even though the binding properties of SpPriA are not correlated with the high or low GC content of DNA substrates ( Figure 3). In addition, we demonstrate that the connecting loop between the 3′ BD and WH motifs is important for SpPriA-DNA binding ability but has limited impact on SpPriA ATPase activity. The primosome accessory protein DnaD stimulates both the ATPase and DNA unwinding activities of SpPriA. Thus, although SpPriA itself is inefficient at unwinding DNA with high GC content, SpDnaD enables the helicase to overcome this restriction during the DNA unwinding reaction.