Molecular insights into replication initiation in a multipartite genome harboring bacterium Deinococcus radiodurans
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100451
ISSN1083-351X
AutoresGanesh Kumar Maurya, Reema Chaudhary, Neha Pandey, Hari S. Misra,
Tópico(s)Bacteriophages and microbial interactions
ResumoDeinococcus radiodurans harbors a multipartite ploid genome system consisting of two chromosomes and two plasmids present in multiple copies. How these discrete genome elements are maintained and inherited is not well understood. PprA, a pleiotropic protein involved in radioresistance, has been characterized for its roles in DNA repair, genome segregation, and cell division in this bacterium. Here, we show that PprA regulates ploidy of chromosome I and II and inhibits the activity of drDnaA, the initiator protein in D. radiodurans. We found that pprA deletion resulted in an increased genomic content and ploidy of both the chromosomal elements. Expression of PprA in trans rescued the phenotypes of the pprA mutant. To understand the molecular mechanism underlying these phenotypes, we characterized drDnaA and drDnaB. As expected for an initiator protein, recombinant drDnaA showed sequence-specific interactions with the putative oriC sequence in chromosome I (oriCI). Both drDnaA and drDnaB showed ATPase activity, also typical of initiator proteins, but only drDnaB exhibited 5′→3′ dsDNA helicase activity in vitro. drDnaA and drDnaB showed homotypic and heterotypic interactions with each other, which were perturbed by PprA. Interestingly, PprA has inhibited the ATPase activity of drDnaA but showed no effect on the activity of drDnaB. Regulation of chromosome copy number and inhibition of the initiator protein functions by PprA strongly suggest that it plays a role as a checkpoint regulator of the DNA replication initiation in D. radiodurans perhaps through its interaction with the replication initiation machinery. Deinococcus radiodurans harbors a multipartite ploid genome system consisting of two chromosomes and two plasmids present in multiple copies. How these discrete genome elements are maintained and inherited is not well understood. PprA, a pleiotropic protein involved in radioresistance, has been characterized for its roles in DNA repair, genome segregation, and cell division in this bacterium. Here, we show that PprA regulates ploidy of chromosome I and II and inhibits the activity of drDnaA, the initiator protein in D. radiodurans. We found that pprA deletion resulted in an increased genomic content and ploidy of both the chromosomal elements. Expression of PprA in trans rescued the phenotypes of the pprA mutant. To understand the molecular mechanism underlying these phenotypes, we characterized drDnaA and drDnaB. As expected for an initiator protein, recombinant drDnaA showed sequence-specific interactions with the putative oriC sequence in chromosome I (oriCI). Both drDnaA and drDnaB showed ATPase activity, also typical of initiator proteins, but only drDnaB exhibited 5′→3′ dsDNA helicase activity in vitro. drDnaA and drDnaB showed homotypic and heterotypic interactions with each other, which were perturbed by PprA. Interestingly, PprA has inhibited the ATPase activity of drDnaA but showed no effect on the activity of drDnaB. Regulation of chromosome copy number and inhibition of the initiator protein functions by PprA strongly suggest that it plays a role as a checkpoint regulator of the DNA replication initiation in D. radiodurans perhaps through its interaction with the replication initiation machinery. The origin of replication in bacterial chromosome (oriC) is a discrete locus that contains AT-rich conserved DNA motifs and a varying number of 9 mer repeats of nonpalindromic sequences called DnaA boxes. These boxes are recognized by a replication initiator protein named DnaA, followed by the assembly of replication initiation complex at oriC (1Messer W. The bacterial replication initiator DnaA. DnaA and oriC, the bacterial mode to initiate DNA replication.FEMS Microbiol. Rev. 2002; 26: 355-374PubMed Google Scholar, 2Mott M.L. Berger J.M. DNA replication initiation: Mechanisms and regulation in bacteria.Nat. Rev. Microbiol. 2007; 5: 343-354Crossref PubMed Scopus (251) Google Scholar). Mechanisms underlying replication initiation have been characterized in large number of bacteria harboring limited copies of single circular chromosome as inheritable genetic material (3Zakrzewska-Czerwińska J. Jakimowicz D. Zawilak-Pawlik A. Messer W. Regulation of the initiation of chromosomal replication in bacteria.FEMS Microbiol. Rev. 2007; 31: 378-387Crossref PubMed Scopus (86) Google Scholar, 4Leonard A.C. Grimwade J.E. The orisome: Structure and function.Front. Microbiol. 2015; 6: 545Crossref PubMed Scopus (59) Google Scholar). In Escherichia coli, it has been shown that DnaA-ATP oligomer binds to DnaA boxes at oriC and unwinds the adjacent AT-rich region. Subsequently, a hexameric complex of replicative helicase DnaB and its loader DnaC (DnaB6-DnaC6) is recruited to the unwound region in oriC resulting in the formation of the prepriming complex (5Skarstad K. Katayama T. Regulating DNA replication in bacteria.Cold Spring Harb. Perspect. Biol. 2013; 5a012922Crossref PubMed Scopus (126) Google Scholar, 6Chodavarapu S. Kaguni J.M. Replication initiation in bacteria.in: The Enzymes. Vol 39. Academic Press, New York, NY2016: 1-30Google Scholar). This provides the site for binding of primase and activation of various events required for the progression of the replication complex that includes DNA polymerase III holoenzyme. 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Recently, the bacteria with multiple copies of the multipartite genome system have been reported. Notably, most of them are either parasites to some forms of life or exhibit super tolerance to abiotic stresses (9Misra H.S. Maurya G.K. Kota S. Charaka V.K. Maintenance of multipartite genome system and its functional significance in bacteria.J. Genet. 2018; 97: 1013-1038Crossref PubMed Scopus (8) Google Scholar). The ploidy of chromosomes in these bacteria allowed us to revisit the mechanism of oriC regulation as known in bacteria containing less than two copies of circular chromosome per cell. Mechanisms underlying the regulation of oriC function in multipartite genome harboring bacteria have not been studied in detail. In the case of Vibrio cholerae, which harbors two chromosomes, namely chromosome I (Chr I) and chromosome II (Chr II), the Chr I replicates similar to the E. coli chromosome while Chr II follows a replication mechanism that is akin to low copy number P1 and F plasmids (10Egan E.S. Fogel M.A. Waldor M.K. Divided genomes: Negotiating the cell cycle in prokaryotes with multiple chromosomes.Mol. Microbiol. 2005; 56: 1129-1138Crossref PubMed Scopus (116) Google Scholar, 11Egan E.S. Waldor M.K. Distinct replication requirements for the two Vibrio cholerae chromosomes.Cell. 2003; 114: 521-530Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 12Jha J.K. Baek J.H. Venkova-Canova T. Chattoraj D.K. Chromosome dynamics in multichromosome bacteria.Biochim. Biophys. Acta. 2012; 1819: 826-829Crossref PubMed Scopus (24) Google Scholar, 13Ramachandran R. Jha J. Paulsson J. Chattoraj D. 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Reassembly of shattered chromosomes in Deinococcus radiodurans.Nature. 2006; 443: 569-573Crossref PubMed Scopus (312) Google Scholar, 17Cox M.M. Battista J.R. Deinococcus radiodurans - the consummate survivor.Nat. Rev. Microbiol. 2005; 3: 882-892Crossref PubMed Scopus (473) Google Scholar, 18Slade D. Radman M. Oxidative stress resistance in Deinococcus radiodurans.Microbiol. Mol. Biol. Rev. 2011; 75: 133-191Crossref PubMed Scopus (428) Google Scholar, 19Misra H.S. Rajpurohit Y.S. Kota S. Physiological and molecular basis of extreme radioresistance in Deinococcus radiodurans.Curr. Sci. 2013; 104: 194-205Google Scholar). It harbors a multipartite genome system comprised of two chromosomes (Chr I (2,648,638 bp) and Chr II (412,348 bp)) and a megaplasmid (177,466 bp) and a plasmid (45,704 bp) (20White O. Eisen J.A. Heidelberg J.F. Hickey E.K. Peterson J.D. Dodson R.J. Haft D.H. Gwinn M.L. Nelson W.C. Richardson D.L. Moffat K.S. Qin H. Jiang L. Pamphile W. Crosby M. et al.Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1.Science. 1999; 286: 1571-1577Crossref PubMed Scopus (750) Google Scholar). Interestingly, each genome element is present in multiple copies per cell (21Hansen M.T. Multiplicity of genome equivalents in the radiation-resistant bacterium Micrococcus radiodurans.J. Bacteriol. 1978; 134: 71-75Crossref PubMed Google Scholar). Chr I of D. radiodurans encodes the putative DnaA (DR_0002) and DnaB (DR_0549) (hereafter named drDnaA and drDnaB, respectively) while chromosome II encodes PprA (DR_A0346), which has been characterized for various functions (22Narumi I. Satoh K. Cui S. Funayama T. Kitayama S. Watanabe H. PprA: A novel protein from Deinococcus radiodurans that stimulates DNA ligation.Mol. Microbiol. 2004; 54: 278-285Crossref PubMed Scopus (123) Google Scholar, 23Kota S. Charaka V.K. Ringgaard S. Waldor M.K. Misra H.S. PprA contributes to Deinococcus radiodurans resistance to nalidixic acid, genome maintenance after DNA damage and interacts with deinococcal topoisomerases.PLoS One. 2014; 9e85288Crossref PubMed Scopus (26) Google Scholar, 24Kota S. Rajpurohit Y.S. Charaka V.K. Satoh K. Narumi I. Misra H.S. DNA gyrase of Deinococcus radiodurans is characterized as type II bacterial topoisomerase and its activity is differentially regulated by PprA in vitro.Extremophiles. 2016; 20: 195-205Crossref PubMed Scopus (11) Google Scholar, 25Adachi M. Hirayama H. Shimizu R. Satoh K. Narumi I. Kuroki R. Interaction of double-stranded DNA with polymerized PprA protein from Deinococcus radiodurans.Protein Sci. 2014; 23: 1349-1358Crossref PubMed Scopus (9) Google Scholar). Recently, the extended structure of PprA has been reported where a possibility of it acting as a protein scaffold has been suggested (26Adachi M. Shimizu R. Shibazaki C. Satoh K. Fujiwara S. Arai S. Narumi I. Kuroki R. Extended structure of pleiotropic DNA repair-promoting protein PprA from Deinococcus radiodurans.FASEB J. 2019; 33: 3647-3658Crossref PubMed Scopus (4) Google Scholar). Here, for the first time, we report the functional characterization of chromosome replication initiation proteins drDnaA and drDnaB in D. radiodurans and demonstrate that PprA plays an important role in regulation of DNA replication. The pprA mutant showed an increased copy number of chromosome I (ChrI) and chromosome II (ChrII), which was complemented by the in trans expression of the wild-type PprA. Interestingly, there was no effect of PprA over expression on copy number of the different genome elements in the wild-type cells. drDnaA was characterized as a sequence-specific origin of replication (oriCI) binding protein and an oriCI responsive ATPase. drDnaB was found to be an ATP-dependent 5′→3′ dsDNA helicase and showed higher affinity for single-stranded DNA (ssDNA) than double-stranded DNA (dsDNA). Interestingly, PprA interacted with drDnaA at a relatively higher affinity than drDnaB and inhibited both homotypic and heterotypic interactions of these proteins. Further, PprA downregulated the ATPase activity of drDnaA but showed no effect on ATPase and helicase activities of drDnaB. These results suggest that drDnaA and drDnaB carry out the necessary functions required for initiation of replication at oriCI in D. radiodurans. Furthermore, the interference imposed by PprA in the physicochemical properties of these replication proteins as well as an increase in the copy numbers of both primary and secondary chromosomes in its absence together suggested the involvement of PprA in regulation of chromosomal replication in this bacterium. Earlier, the regulatory role of PprA in cell division and genome maintenance has been demonstrated (23Kota S. Charaka V.K. Ringgaard S. Waldor M.K. Misra H.S. PprA contributes to Deinococcus radiodurans resistance to nalidixic acid, genome maintenance after DNA damage and interacts with deinococcal topoisomerases.PLoS One. 2014; 9e85288Crossref PubMed Scopus (26) Google Scholar, 24Kota S. Rajpurohit Y.S. Charaka V.K. Satoh K. Narumi I. Misra H.S. DNA gyrase of Deinococcus radiodurans is characterized as type II bacterial topoisomerase and its activity is differentially regulated by PprA in vitro.Extremophiles. 2016; 20: 195-205Crossref PubMed Scopus (11) Google Scholar, 27Devigne A. Mersaoui S. Bouthier-de-la-Tour C. Sommer S. Servant P. The PprA protein is required for accurate cell division of γ-irradiated Deinococcus radiodurans bacterium.DNA Repair. 2013; 12: 265-272Crossref PubMed Scopus (29) Google Scholar, 28Devigne A. Guérin P. Lisboa J. Quevillon-Cheruel S. Armengaud J. Sommer S. Bouthier de la Tour C. Servant P. PprA protein is involved in chromosome segregation via its physical and functional interaction with DNA gyrase in irradiated Deinococcus radiodurans bacteria.mSphere. 2016; 1e00036-15Crossref PubMed Scopus (15) Google Scholar). In this study, the DNA content and the copy number of genome elements in pprA deletion mutant were compared with the wild-type D. radiodurans. The total DNA content in mutant cells (∼8.41 ± 1.05 fg per cell) was found to be approximately threefold higher than wild-type cells (∼3.05 ± 0.47 fg per cell) (Fig. 1A). Similarly, the DAPI fluorescence in ΔpprA mutant was approximately twofold higher than that of wild type (Fig. 1B). The cell scan analysis of DAPI stained cells showed that ∼80% of ΔpprA cells have approximately twofold higher DAPI fluorescence as compared with wild-type cells grown identically (Fig. 1C). When we checked the copy number of each replicon per cell, we found that the average copy number of Chr I and Chr II was ∼2.5- to 3-fold higher in ΔpprA mutant as compared with the wild type. For instance, the average copy number of Chr I was ∼8 per cell in wild type while it was ∼18 per cell in the ΔpprA mutant. The average copy number of Chr II also increased from ∼7 in wild type to ∼17 per cell in the ΔpprA mutant (Fig. 1D). Further we checked the effect of in trans expression of PprA on the copy number of these genome elements in wild type and ΔpprA mutant. We observed nearly no effect of PprA over expression in wild type, whereas the ΔpprA mutant expressing PprA in trans could restore the wild-type copies of the genome element (Fig. 1D). Interestingly, the copy number of megaplasmid and small plasmid did not change under any of these conditions. These results indicated a possible role of PprA in the maintenance of the chromosome copy number possibly by regulating the replication of DNA in D. radiodurans. To obtain the mechanistic insights into the regulatory role of PprA in DNA replication, the physical and functional interaction of PprA with drDnaA or drDnaB proteins was monitored using bacterial two-hybrid system. For this, T18-tagged PprA and T25-tagged drDnaA or drDnaB were coexpressed in E. coli BTH101 (cyaA−). Interaction of two target proteins tagged with T18 and T25 domains of CyaA would reconstitute the active CyaA resulting in induction of transcription from β-galactosidase gene in BTH101. Expression of β-galactosidase as an indication of protein–protein interaction was monitored using spot test. PprA showed interaction with both drDnaA and drDnaB as indicated by a blue color in the spot test (Fig. 2A). The interaction of PprA with these proteins was further checked using surface plasmon resonance (SPR) using purified recombinant drDnaA or drDnaB (Fig. S1). Results showed a concentration-dependent increase in the SPR signals for both drDnaA and drDnaB (Fig. 2, B and C). The dissociation constant (Kd) for drDnaA was 5.41 × 10−7 ± 1.8 X 10−8 [M] while it was 9.71 × 10−7 ± 1.1 × 10−8 [M] for drDnaB indicating that PprA interacts with drDnaA with approximately twofold higher affinity than with drDnaB. Interaction of C-terminal deletion (CTD) mutant of drDnaA (DnaAΔCt) with PprA was tested in surrogate E. coli using co-immunoprecipitation. Results showed that the deletion of CTD did not affect drDnaA interaction with PprA (Fig. 2D) suggesting that drDnaA might interact with PprA through its N-terminal domain and/or the middle region of the protein. In vivo interaction of PprA with drDnaA and dDnaB was tested using co-immunoprecipitation assays with the cellular protein extracts from D. radiodurans cells transformed with T18-tagged drDnaA or drDnaB expressing plasmid. Endogenous PprA was precipitated using anti-PprA antibodies and the presence of T18-tagged drDnaA/drDnaB was probed using immunoblotting with T18 antibodies. Consistent with the above results, both drDnaA and drDnaB had co-immunoprecipitated with PprA in vivo (Fig. 2E). These results together suggested that PprA interacts with both drDnaA and drDnaB of D. radiodurans. The putative origin of replication of chromosome I (oriCI) in D. radiodurans R1, spanning 1183 to 1903 bp upstream of drdnaA gene (DR_0002), was predicted using the DOriC database (DoriC accession number – ORI10010007) (29Luo H. Gao F. DoriC 10.0: An updated database of replication origins in prokaryotic genomes including chromosomes and plasmids.Nucleic Acids Res. 2018; 47: D74-D77Crossref Scopus (30) Google Scholar). Sequence analysis using WebLogo online tool revealed the presence of consensus sequences of 13 copies of 9-mer DnaA-boxes (30Crooks G.E. Hon G. Chandonia J.M. Brenner S.E. WebLogo: A sequence logo generator.Genome Res. 2004; 14: 1188-1190Crossref PubMed Scopus (7179) Google Scholar) between 1273 and 1772 bp (∼500 bp) upstream of the drdnaA gene. A 46.2% GC content of oriCI indicated an AT-rich sequence, typical of canonical oriC sequences. The structure of oriCI was found to be different as compared with the E. coli oriC. It is comparatively longer than the E. coli oriC and contains only eight out of 13 perfect E. coli like DnaA boxes (TTATCCACA). Five out of 13 DnaA boxes were imperfect with single nucleotide difference from the E. coli type boxes and were similar to other bacteria such as Cyanothece 51142, Thermus thermophilus, and Bacillus subtilis (Fig. S2) (31Huang H. Song C.C. Yang Z.L. Dong Y. Hu Y.Z. Gao F. Identification of the replication origins from cyanothece ATCC 51142 and their interactions with the DnaA protein: From in silico to in vitro studies.Front. Microbiol. 2015; 6: 1370Crossref PubMed Scopus (4) Google Scholar, 32Schaper S. Nardmann J. Lu-der G. Lurz R. Speck C. Messer W. Identification of the chromosomal replication origin from Thermus thermophilus and its interaction with the replication initiator DnaA.J. Mol. Biol. 2000; 299: 655-665Crossref PubMed Scopus (26) Google Scholar, 33Moriya S. Fukuoka T. Ogasawara N. Yoshikawa H. Regulation of initiation of the chromosomal replication by DnaA-boxes in the origin region of the Bacillus subtilis chromosome.EMBO J. 1988; 7: 2911-2917Crossref PubMed Scopus (47) Google Scholar). A web logo of these DnaA boxes was created yielding a consensus sequence of T(A/G)TA(T)TCCACA. These DnaA boxes are distributed randomly on both sense (four DnaA boxes) and antisense (nine DnaA boxes) strands of chromosome I. This suggested that oriCI in chromosome I of D. radiodurans is largely similar to oriC of E.coli with some deviations. Whether these changes have any functional significance, in the context of regulation of replication initiation, needs to be explored. Binding of recombinant drDnaA (Fig. S1A) to [32P] end labeled oriCI containing 13 repeats of DnaA-boxes was checked by electrophoretic mobility shift assays (EMSA). The recombinant drDnaA showed sequence-specific interaction with oriCI as the binding did not change even in the presence of 50-fold excess of cold nonspecific DNA (Fig. 3, A and B). Interestingly, the binding affinity of drDnaA for oriCI (Kd = 1.68 ± 0.23 μM) was increased by approximately eightfold in presence of ATP (Kd = 0.243 ± 0.02 μM) (Fig. 3, C and D). Further, binding of [32P] end labeled nonspecific DNA with increasing concentrations of drDnaA was displaced by addition of higher molar concentration of cold oriCI DNA confirming the sequence-specific interaction of drDnaA to oriCI (Fig. 3E). In addition, the binding affinity of drDnaA for oriCI decreased upon reducing the number of DnaA boxes (Fig. S3) in the presence of ATP (Table 1). The effect of histidine tag on DNA-binding activity drDnaA was ruled out as both (his)6 tagged and non-(his)6 tagged drDNA showed nearly similar Kd for dsDNA (Fig. S4). Similar to E. coli DnaA, which shares 44.4% identity and 72% similarity with drDnaA (data not shown), drDnaA showed specific binding even for the oriCI sequence with only a single DnaA box. However, the affinity of drDnaA for a perfect DnaA box (TTATCCACA) was approximately twofold higher (Kd = 2.88 ± 0.28 μM) than the affinity (Kd = 4.21 ± 0.15 μM or 4.36 ± 0.18 μM) for imperfect DnaA boxes (TTTTCCACA or GTATCCACA) (Table 1). These results suggested that drDnaA binds to oriCI in a sequence-specific manner and this interaction is stimulated by ATP.Table 1Dissociation constant (Kd) of drDnaA with oriCI and its repeat variants with the different number of DnaA boxes was measured in the presence and absence of ATPNumber of repeatsLength (bp)ATPDissociation constant (Kd)Mean ± SD (μM)Full length (13 repeats)500 bp−1.78 ± 0.23Full length (13 repeats)500 bp+0.243 ± 0.02111 Repeats261 bp+0.241 ± 0.017 Repeats164 bp+0.373 ± 0.013 Repeats37 bp+1.08 ± 0.121 Repeat (TTATCCACA)16 bp+2.88 ± 0.281 (TTTTCCACA) nonperfect repeat16 bp+4.21 ± 0.151 (GTATCCACA) nonperfect repeat16 bp+4.36 ± 0.18Recombinant purified drDnaA was incubated with radiolabeled repeat variants of linear oriCI in the presence (+) and absence (−) of ATP and EMSA was carried out, and autoradiograms were developed as shown in Figure 4. Fractions of DNA bound to protein were estimated densitometrically and plotted as a function of protein concentration. The Kd for the curve fitting of individual plots was determined using GraphPad Prism6 software. Open table in a new tab Recombinant purified drDnaA was incubated with radiolabeled repeat variants of linear oriCI in the presence (+) and absence (−) of ATP and EMSA was carried out, and autoradiograms were developed as shown in Figure 4. Fractions of DNA bound to protein were estimated densitometrically and plotted as a function of protein concentration. The Kd for the curve fitting of individual plots was determined using GraphPad Prism6 software. Since, drDnaA showed a relatively higher affinity for oriCI in the presence of ATP (Fig. 3C), ATP hydrolysis by drDnaA in the presence and absence of oriCI was tested. Results showed that drDnaA hydrolyzes [32P] αATP into [32P] αADP (Fig. 4A). Interestingly, the ATP hydrolysis by drDnaA is stimulated in the presence of oriCI (Fig. 4, B and C) but not in the presence of nonspecific dsDNA (Fig. S5). The helix-turn-helix motif in domain IV at the C terminal of DnaA is known to confer sequence specificity for DnaA–oriC interaction in many bacteria (34Roth A. Messer W. The DNA binding domain of the initiator protein DnaA.EMBO J. 1995; 14: 2106-2111Crossref PubMed Scopus (122) Google Scholar, 35Sutton M.D. Kaguni J.M. The Escherichia coli dnaA gene: Four functional domains.J. Mol. Biol. 1997; 12: 546-561Crossref Scopus (62) Google Scholar, 36Majka J. Jakimowicz D. Messer W. Schrempf H. Lisowski M. Zakrzewska-Czerwinska J. Interactions of the Streptomyces lividans initiator protein DnaA with its target.Eur. J. Biochem. 1999; 260: 325-335Crossref PubMed Scopus (30) Google Scholar, 37Blaesing F. Weigel C. Messer W. Analysis of the DNA binding domain of Escherichia coli DnaA protein.Mol. Microbiol. 2000; 36: 557-569Crossref PubMed Scopus (74) Google Scholar, 38Messer W. Blaesing F. Majka J. Nardmann J. Schaper S. Schmidt A. Seitz H. Speck C. Tüngler D. Wegrzyn G. Weigel C. Welzeck M. Zakrzewska-Czerwinska J. Functional domains of DnaA proteins.Biochimie. 1999; 81: 819-825Crossref PubMed Scopus (78) Google Scholar, 39Fujikawa N. Kurumizaka H. Nureki O. Terada T. Shirouzu M. Katayama T. Yokoyama S. Structural basis of replication origin recognition by the DnaA protein.Nucleic Acids Res. 2003; 31: 2077-2086Crossref PubMed Scopus (138) Google Scholar). Therefore, the binding of drDnaA having a deleted C terminal (domain IV) (DnaAΔCt) (Fig. 5A) with oriCI was checked in the presence and absence of ATP. DnaAΔCt failed to bind oriCI (Fig. 5B) and the ATPase activity of DnaAΔCt (Fig. 5C) did not change in the presence of oriCI (Fig. 5, D and E). Further, the effect of CTD deletion on rate of ATP hydrolysis was calculated using both drDnaA and DnaAΔCt proteins at 30 nM [32P] αATP. The rate of ATP hydrolysis in drDnaA (0.76 ± 0.04 nM/min) was found to be nearly similar to DnaAΔCt (0.81 ± 0.05 nM/min). These results suggested that the C-terminal domain IV of drDnaA is essential for its binding to oriCI and thus for oriCI-dependent stimulation of ATPase function but seems to have no role in the ATPase activity of drDnaA per se.Figure 5Role of C-terminal putative helix-turn-helix motifs containing domain in drDnaA function. The 99 amino acids of drDnaA were removed from its C terminal and resulting DnaAΔCt derivative was generated (A). An increasing concentration of recombinant protein was incubated with radiolabeled linear oriCI (oriI) DNA as described in Figure 4A. Products were analyzed on native PAGE and autoradiogram was developed (B). Similarly, 2 μM concentration of DnaAΔCt was incubated for different time points with [32P]-αATP in the absence (C) and presence (D) of linear oriCI (oriI) DNA. Products were separated on TLC and analyzed as described in Figure 4. The percentage of ADP/ATP ratios was plotted as a function of time (E). Results were analyzed using Student's t-test and significant difference in data sets with p values of 0.05 or less is shown as (∗).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Binding of recombinant drDnaB to the oriCI was checked by EMSA. drDnaB showed nonspecific binding to oriCI as chase with nonspecific dsDNA significantly competed out oriCI bound to drDnaB. The affinity of drDnaB for oriCI (Kd = 8.89 ± 0.92 μM) did not change in the presence of ATP (Kd = 8.23 ± 0.41 μM). Interestingly, drDnaB showed approximately threefold higher affinity to ssDNA substrate (Kd = 3.11 ± 0.11 μM) as compared with dsDNA (oriCI) (Kd = 8.89 ± 0.92 μM). Further, binding of drDnaB to ssDNA increased by nearly twofold in the presence of ATP (Kd = 1.42 ± 0.05 μM) (Fig. 6) and approximately threefold in the presence of ATPγS (1.02 ± 0.06 μM) (Fig. S6). Addition of ATP increased drDnaB affinity for ssDNA but not for dsDNA. These results suggested that drDnaB preferentially binds to ssDNA over dsDNA both in the presence and absence of ATP. Similar to drDnaA, drDnaB could hydrolyze [32P]-αATP to [32P]-αADP (Fig. 7A). The stimulatory effect of ssDNA on ATP hydrolysis was observed albeit at a very low level and only at the lower ATP to protein ratios (Fig. 7, B and C). Similar observations were reported earlier for other DnaB homologs (40Biswas E.E. Barnes M.H. Moir D.T. Biswas S.B. An essential DnaB helicase of Bacillus anthracis: Identification, characterization, and mechanism of action.J. Bacteriol. 2009; 191: 249-260Crossref PubMed Scopus (7) Google Scholar, 41Zhang H. Zhang Z. Yang J. He Z.G. Functional characterization of DnaB helicase and its modulation by single-stranded DNA binding protein in Mycobacterium tuberculosis.FEBS J. 2014; 281: 1256-1266Crossref PubMed Scopus (12) Google Scholar). These results suggested that drDnaB is an ssDNA-binding ATPase and its binding to ssDNA is affected by ATP hydrolysis in vitro.Figure 7ATPase activity of purified recombinant drDnaB. An increasing concentration of drDnaB (DnaB) was incubated with [32P]-αATP (αATP) in the absence (A) and presence (B) of ssDNA and generation of [32P]-αADP (αADP) product was detected on TLC. Spot intensity was quantified densitometrically and the percent of ADP/ATP ratios wasplotted as mean ± SD (n = 3) (C). Results were analyzed using Student's t-test and a significant difference in data set with p values o
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