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J. Biol. Chem., Vol. 282, Issue 27, 19917-19927, July 6, 2007
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12
From the
Genome Dynamics Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, the Department of Structural Biology, Biomolecular Engineering Research Institute, Suita, Osaka 565-0874, and the ¶Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan
Received for publication, March 2, 2007 , and in revised form, April 20, 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Escherichia coli PriA protein was originally identified as a component essential for conversion of the 
X174 single-stranded genome to double-stranded replicative form. PriA binds to n'-pas (n'-primosome assembly site; a single-stranded DNA hairpin structure recognized by PriA protein) and assembles a primosome, a complex responsible for primer RNA synthesis and duplex unwinding at a replication fork (6, 7). Genetic analyses of priA mutants have suggested that PriA plays crucial roles in cellular responses to replication fork arrest. PriA null cells exhibit severe sensitivity to DNA damages including UV and mitomycin C (8, 9), and are defective in RecA-dependent stable DNA replication induced by replication fork blocks (10). Indeed, PriA can specifically recognize and bind to D-loop like and arrested fork structures in vitro (11-13) and establishes a primosome on these structures (14-17). Other biochemical activities of PriA protein include ATP hydrolysis that strictly depends on binding to specific DNAs (7, 18) and 3' to 5' DNA helicase actions (19, 20).
PriA is composed of two domains, namely the N-terminal DNA binding domain and the C-terminal DEXH-type helicase domain (21-23). The former contains a 3' terminus binding pocket that specifically recognizes and binds to a 3'-end of DNA. The presence of this 3'-end binding structure facilitates high affinity and stable binding to D-loop or arrested fork structures carrying a 3'-end at the branch point (24).
PriA is a vital protein for cellular responses to replication fork blocks in bacterial cells, and it may serve as a sensor and stabilizer for the arrested replication forks (25-27). Therefore, it would be very important to dissect the architecture of PriA protein bound to the arrested forks and other substrates to elucidate the mechanism by which this protein recognizes blocked replication forks. It is also an intriguing question, which domain of PriA is required for activation of ATP hydrolysis.
To address these issues, we have conducted detailed analyses of protein-DNA complexes and ATPase activation using various DNA substrates and mutant PriA proteins. The results indicate that the PriA binds to DNA in two distinct modes, either dependent on or independent of the 3' terminus recognition. In the former, PriA makes a stable, bipartite interaction at the fork junction. The N-terminal DNA binding domain contains a module that may recognize the duplex to single-strand DNA junction, in addition to the 3' terminus binding pocket. DNA binding through either module can stimulate ATP hydrolysis by PriA, and engagement of the both modules leads to the most stable binding and maximum activation of the ATPase activity.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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DNA Substrates—Forked or extension substrates were constructed by annealing the oligonucleotides as indicated in supplemental Table S1. Substrates designated [3'-P] carries the leading strand whose 3'-end is phosphorylated. The 5'-end of the labeled oligonucleotide was phosphorylated by T4 polynucleotide kinase (New England Biolabs) and [
-32P]ATP (GE Healthcare) prior to annealing. Each substrate was isolated from the polyacrylamide gel as described previously (22).
Nuclease Protection Assay—Reaction was performed in a mixture (25 µl) containing 40 mM Hepes/KOH (pH 7.6), 8 mM magnesium acetate, 0.5 mM CaCl2, 40 mM potassium glutamate, 20 µg/ml bovine serum albumin, 0.8-3 nM substrate, and 4-64 nM PriA full-length protein as indicated in the figures. For other polypeptides, the concentrations are shown in the figures. To prevent aggregation of the polypeptides in binding assays, 20-30 mM CHAPSO3 was added in the reactions, where indicated. After incubation for 20 min at 30 °C, 0.001 unit of DNase I (TAKARA), 0.04-0.9 units of P1 nuclease (Roche), or 50 units of exonuclease III (New England Biolabs) were added to the reaction and incubation was continued for 1 min, followed by addition of an equal volume of 2x stop solution containing 20 mM EDTA, 1% SDS, 200 mM NaCl, and 125 µg/ml yeast tRNA. Digested fragments were extracted by phenol and recovered by ethanol precipitation before being resolved in 80% formamide. After heating at 96 °C for 5 min, each sample was applied to 10 or 12% denaturing polyacrylamide sequencing gels (40 cm) containing 8 M urea. Electrophoresis was conducted in 0.5 x Tris borate EDTA at 40 W constant electric power for 1 h. Digestion patterns were visualized by autoradiography.
ATPase Assay—ATP hydrolyzing activity of the wild-type or mutant PriA protein, N- or C-terminal polypeptide was measured by thin layer chromatography as previously described (22). Reactions were performed in a 12.5-µl mixture containing 40 mM Hepes/KOH (pH 7.6), 8 mM magnesium acetate, 40 mM potassium glutamate, 1 mM dithiothreitol, 10% glycerol, and effector DNA. Protein titration was performed as indicated in the figures. Each reaction was started by addition of 0.2 mM ATP with 0.5 µCi of [-32P]ATP and incubation was conducted at 30 °C with or without 4 nM effector DNAs. After a 20-min incubation, 1-µl samples were withdrawn and spotted on the polyethyleneimine cellulose thin layer chromatography plate (Merck). Dried spots were developed by 0.5 M LiCl and 1 M formic acid. Each plate was autoradiographed. Free [32P]phosphate was quantified by Fuji Photo Film Multi Gauge (version 3.0) software and values of hydrolyzed ATP were plotted against protein concentrations.
BIAcore Analyses of DNA Binding—BIAcore analyses were conducted as described previously (24). The amount of the immobilized oligonucleotide is indicated in the figure as resonance units. Sequence of the oligonucleotide for A-fork [3'] mimic (AFM) is listed in supplemental Table S1. For AFM [3'-P], the 3'-end of the oligonucleotide was phosphorylated as described above. The dissociation constants were determined by a global fitting method using the BIA evaluation software 3.1.
Gel Shift Assay—Reactions were conducted as described previously (22). Gels were dried and autoradiographed. DNA binding activity of PriA-(109-181) was assayed by mixing the polypeptide (at 50 µM) with a substrate DNA (at 10 µM). After incubation for 10 min at room temperature, glycerol was added at a final concentration of 5% and samples were directly applied to 0.7% agarose gel, followed by electrophoresis with 50 V constant voltage for 40 min at 4 °C in 1x Tris borate buffer. Gel was stained with ethidium bromide and photographed under UV illuminator.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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With increasing amounts of full-length PriA protein, two areas were found to be protected; one centered on the branch point (-12 to +10) and the other from -22 to -16 within the unreplicated duplex region. A hypersensitive site was detected at +10 in the leading strand arm (Fig. 1B, lanes 1-8). When PriA-(1-193) was used, weak protection was detected near the branch point through the leading strand arm at lower concentrations (80-160 nM) of the polypeptide, but the entire segment was protected at higher concentrations (Fig. 1B, lanes 9-18). This is probably due to aggregation of PriA-(1-193) on the substrate, because the same complexes do not enter the gel at PriA concentrations above 320 nM in gel shift assays. We found that addition of the zwitterionic detergent CHAPSO at 20 mM reduced the formation of these aggregates (supplemental Fig. S1B). Therefore, we next conducted DNase I footprinting in the presence of 20 mM CHAPSO. Under this condition, nonspecific binding was significantly reduced even at high concentrations of the polypeptide, and weak protection near the branch point and over the leading strand arm (+4to +9 and -3to -2) was observed (Fig. 1B, lanes 19-26). However, we did not observe significant protection on the unreplicated duplex region, which was seen with the full-length PriA. We next conducted exonuclease III protection assays. In this assay, a protein bound to DNA blocks processive movement of the nuclease, revealing the 3'-extent of the binding. On A-fork [3'], strong blocks were detected with the full-length PriA at +4to +8 on the leading strand arm and at -4, -14, and -26 on the unreplicated arm (Fig. 1C, lanes 3 and 4 and lanes 10 and 11, respectively). This is consistent with the result of the DNase I footprinting assay (Fig. 1B, lanes 1-8). With PriA-(1-193), blocks around -4 were detected but not those farther away from the junction (Fig. 1C, lane 13). Blocks on the leading strand arm were not obvious (Fig. 1C, lanes 5 and 6). These results are consistent with the notion that PriA binds to a fork carrying a 3' terminus at the branch through bipartite interactions, and that the interactions on the unreplicated arm and on the leading strand arm are largely due to the helicase domain and to the N-terminal DNA binding domain, respectively.
PriA Binds to Fork Structures in Both 3' Terminus-dependent and -independent Manners—We next conducted the DNase I protection assays on other A-fork substrates. The segment (-9 to +8) spanning over the branch point of A-fork [3', 5'] was protected as was seen with A-fork [3'] (Fig. 1D). However, the protection within the unreplicated arm was not as localized as that seen on A-fork [3']. The hypersensitive site detected at +10 on A-fork [3'] was not seen on A-fork [3', 5']. Protection extended over the 13-bp segment in the leading strand arm as more PriA was added. Strong protection was also detected on the lagging strand arm of A-fork [3', 5'](+7to +1, solid line; Fig. 1E, lanes 9-16). A second, weak protection (+17 to +8; dashed line) was also detected on this arm of A-fork [3', 5']. In contrast, interaction of PriA with the lagging strand arm on A-fork [3'] was limited. P1 nuclease protection assays on A-fork [3'] showed very weak protection of only 7 nucleotides from the junction (+1to +7, Fig. 1E, lanes 17-24). On this strand of A-fork [3', 5'], rather strong protection in the unreplicated segment was also detected, and the protected segment extended as more PriA was added (up to -17, Fig. 1E, lanes 9-16). On A-fork [5'], feeble protection was detected on the unreplicated arm near the branch point (-1to -6), which extended over the larger segment on both the lagging strand and unreplicated arms, when more PriA was added (Fig. 1E, lanes 1-8), consistent with the lower affinity of PriA to this substrate in gel shift assays (12, 24). Y-fork structure, to which PriA binds with 1 order of magnitude lower affinity than to A-fork [3'] in the gel shift assay (data not shown), was only very weakly protected from DNase I digestion on both leading and lagging strand templates (Fig. 1F). Weak periodic protection (alternate appearance of protected and non-protected regions) was detected on the duplex arm as well. We also conducted P1 nuclease footprinting assays on Y-fork 49 to probe interactions on the single-stranded arms more precisely. A 49-mer Y-fork was used to reduce the possibility of a secondary structure in the single-stranded DNA segments. Significant protection was not detected on both arms in this assay (Fig. 1G), indicating that interaction between PriA and single-stranded segments, if any, is weak and maybe transient. These results may indicate that PriA interacts with Y-fork at multiple sites in a rather scattered and transient manner. The bindings of PriA to A-fork [5'] and Y-fork do not involve the 3' terminus and these binding modes appear to be different from that on fork structures carrying a 3' terminus in that the former does not give strongly localized protection.
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Arrested Fork DNA Activates PriA ATPase Activity through Both 3' Terminus-dependent and -independent Interactions—PriA is a DNA-dependent ATPase, and ATP hydrolysis by PriA strictly depends on its binding to DNA (7). We previously reported that a D-loop like structure strongly stimulated ATPase activity of PriA (29). Similarly, A-fork [3'] DNA significantly activated PriA-mediated ATP hydrolysis (Fig. 3, A and C), and the phosphorylation of the 3'-end of the arrested leading strand reduced the level of ATP hydrolysis (Fig. 3, B and C). We also used A-fork [3'] carrying phosphorylated 3'-ends at duplex ends in ATPase assays. On this template as well, phosphorylation of the 3'-end present at the junction (A-fork [3'-P]) reduced the level of ATP hydrolysis with the wild-type PriA, albeit to a lesser extent (Fig. 3D). The L12G,F16G,Y18G mutant PriA (LFY), in which the 3' terminus binding pocket is specifically disrupted by the amino acid substitutions of the critical residues (24), showed an identical level of ATP hydrolysis with A-fork [3'] or A-fork [3'-P] (Fig. 3C), suggesting that 3' terminus-independent interaction also contributes to the stimulation of ATP hydrolysis with this effector DNA. The C-terminal helicase domain (PriA-(194-732)) exhibits only a basal ATP hydrolysis activity, whereas the N-terminal fragment, PriA-(1-193), is completely defective in ATP hydrolysis (Fig. 3E), suggesting that the N-terminal DNA binding domain suppresses the basal ATP hydrolysis activity of the C-terminal domain in the absence of effector DNA, as discussed previously (23). Basal helicase activity of the C-terminal helicase domain polypeptide was not further stimulated by the presence of DNA (Fig. 3, A and E, Fig. 4, A and C). Thus, ATP hydrolysis by PriA may be conducted through coordinated conformational changes of the N-terminal DNA binding and C-terminal helicase domains.
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The N-terminal DNA Binding Segment, PriA-(1-193), May Contain an Additional Domain Recognizing a Feature in the Forked Structure—The ability of A-fork [3'] to activate PriA ATPase activity in the absence of 3' terminus interaction suggests the presence of an additional domain involved in interaction with structured DNAs (Fig. 3, C and D). Because the N-terminal 193- or 181-amino acid polypeptide can bind to 3'-extension structures that do not carry a 3' terminus at the single to duplex junction (Fig. 5A and data not shown), the predicted DNA recognition domain should be present on this polypeptide. Note that there is no significant functional difference between PriA-(1-193) and PriA-(1-181) polypeptides (supplemental Fig. S1). It is most likely that this domain spans the segment 106 to 181. We then compared the binding properties of the two polypeptides, namely PriA-(1-105) and PriA-(1-181) in BIAcore assays. As reported previously, binding of PriA-(1-105) and PriA-(1-181) to a 17-mer oligonucleotide with a free 3'-end could be detected in the BIAcore assays (Kd, 20 and 4.9 µM, respectively), although the 3' phosphorylated 17-mer could not be detected under the same condition (Fig. 5B) (24). This suggests that a 3'-end is the only determinant for the observed binding between the 17-mer and these polypeptides and that the 3' terminus binding pocket is present on the PriA-(1-105) polypeptide (24, 31). To measure binding of these polypeptides to fork-like structures in BIAcore assays, we then developed a novel ligand, "A-fork [3'] mimic" (AFM; Fig. 5C), which self-generates a secondary structure mimicking A-fork [3']. In contrast to the 17-mer, the PriA-(1-181) bound to the AFM with 1 order of magnitude higher affinity (Kd, 390 nM, Fig. 5, C and D), and this binding was still detected even when the 3'-end of the ligand was phosphorylated, although the affinity was considerably decreased (Kd, 4.2 µM, Fig. 5, C and D). The PriA-(1-105) polypeptide, on the other hand, did not show significant binding to AFM under the same condition (Fig. 5, C and D).
We also compared the DNA binding of PriA-(1-193) and PriA-(1-105) in gel shift assay. In this assay, we used A-fork [3'] 49 (P) and Y-fork 49 (P) templates in which free 3'-ends were phosphorylated to specifically detect the binding at the fork structure. PriA-(1-193) could bind not only to the A-fork [3'] but also to Y-fork (Fig. 5E, left panel) with significant affinity. On the other hand, PriA-(1-105) bound to the A-fork [3']ata high concentration, but barely bound to the Y-fork at the same concentration (Fig. 5E, right panel). This result indicates that the presence of the 106-193 segment specifically facilitates binding to the Y-fork structure.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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3' Terminus-independent Mode of PriA Binding to the Arrested Fork Structures—In contrast, on the A-fork [5'], only the 3' terminus-independent mode of binding occurs. This may be manifested by low affinity and extensive and weak interaction of PriA with the lagging strand template as well as with the unreplicated duplex arm (Fig. 1E). This "loose" interaction leads to unreplicated duplex unwinding as well as to displacement of the nascent lagging strand (27) (Fig. 6, modes II and III). PriA may bind to the 3'-extension in a similar manner, displacing the hybridizing DNA (11, 27).
This 3' terminus-independent mode of binding also operates when PriA interacts with Y-fork. PriA may interact with the single-stranded arms as well as with the unreplicated duplex arm, but this interaction appears to be very weak judged from the nuclease protection assays (Fig. 1, F and G). The exonuclease III assays show blocks around -5 (on the unreplicated arm), suggesting the interaction at the fork branch point (supplemental Fig. S5 and Fig. 6, mode VI). PriA is able to displace the Y-fork presumably through translocation on the leading strand template (11, 27, 32). Under this condition on the Y-fork, the helicase domain of PriA would be oriented in a direction opposite to that on the A-fork [3'] (Figs. 1, B and F, and 6, compare modes I and VI). This particular mode of binding would also occur with A-fork [5'] (Figs. 1E and 6, mode III), facilitating the displacement of the unreplicated duplex (27, 32), but does not occur with A-fork [3'] or A-fork [3', 5'], because PriA cannot displace the unreplicated duplex on these substrates (27).
PriA binds to A-fork [3', 5'] with affinity similar to A-fork [3'], suggesting that the binding is dependent on a 3' terminus. The protection on the unreplicated arm was less intense and somewhat more extensive protection was observed on the leading strand arm at higher concentrations of PriA in A-fork [3', 5'] than observed in A-fork [3']. The hypersensitive site appearing at +10 on the leading strand arm in A-fork [3'] was not detected on A-fork [3', 5'] (Fig. 1, B and D), consistent with less localized binding of PriA on the unreplicated arm. In contrast, significant protection was observed on the lagging strand arm on A-fork [3', 5'] (Fig. 1E). P1 nuclease footprinting on A-fork [3'] indicated very little protection on the lagging strand arm (Fig. 1E), indicating that PriA does not interact with the single-stranded lagging strand arm of A-fork [3']. These results show that the mode of interaction of PriA with A-fork [3', 5'] is not identical to that with A-fork [3']. We speculate that PriA interacts with A-fork [3', 5'] in two different orientations in a manner dependent on a 3' terminus (Figs. 1, D and E, 6, modes IV and V). The high affinity binding of PriA to a fork requires interaction of the helicase domain with the unreplicated duplex segment as well. We predict that, on A-fork [3', 5'], the unreplicated arm and lagging strand arm may be equivalent relative to the 3' terminus, generating two different complexes depending on which duplex arm the helicase domain interacts. Thus, the footprints on A-fork [3', 5'] are the sum of these two different interactions (Fig. 1, D and E).
A Second DNA Recognition Module within the N-terminal DNA Binding Domain—Because the N-terminal 193-amino acid polypeptide can bind to A-fork [3'-P] or 3'-extension (supplemental Fig. S6), the domain required for 3' terminus-independent binding is expected to be contained within this polypeptide. Although the N-terminal 105-amino acid polypeptide binds to the 3'-end in the BIAcore assay, it cannot bind to the A-fork [3'] mimic with a phosphorylated 3'-end with appreciable binding constant (Fig. 5, C and D). In contrast, the N-terminal 181-amino acid polypeptide can bind to this ligand even when its 3'-end is phosphorylated. In gel shift assays, PriA-(1-193) can bind to A-fork [3'] as well as to Y-fork, whereas PriA-(1-105) binds to A-fork [3'] but not to Y-fork (Fig. 5E). Thus, the segment 106-181 may likely contain a domain required for 3' terminus-independent recognition.
This second recognition domain within the N-terminal DNA binding domain is likely to recognize the fork structure or duplex to single-stranded DNA junction present in the fork or in the 3'-extension structures. The ability of the N-terminal polypeptide to bind to the 3'-phosphorylated 3'-extension structure (which does not contain a free 3'-end except at the duplex end; supplemental Fig. S6) indicates that the additional domain may recognize the junction from duplex to single strand. Consistent with this, the PriA footprints are detected mainly over the single to double strand transition area on the 3'-extension substrate as well (Fig. 2). We also showed that the isolated polypeptide of PriA-(109-181) can bind to extension structures in vitro (supplemental Fig. S4). These results suggest an intriguing possibility that the recognition of the arrested fork structure by PriA is achieved by concurrent actions of two DNA recognition modules in the N-terminal DNA binding domain, one recognizing the 3' terminus of the leading strand and the other the single to duplex junction.
Possible Modes of ATPase Activation by PriA Protein—PriA hydrolyzes ATP in a manner dependent on its binding to DNA (7). The helicase domain alone exhibits low but constitutive levels of ATPase activity, which is not further activated by addition of DNA (Figs. 3 and 4). Thus, the N-terminal DNA binding domain suppresses the basal ATP hydrolysis activity of the helicase domain, as reported previously (23). DNA binding at the N-terminal segment may induce an allosteric conformational change, activating the ATP hydrolysis activity.
Non-structured single-stranded DNA can also activate ATP hydrolysis by PriA in the absence of single-stranded DNA-binding protein (29). This activation is largely dependent on the presence of a 3' terminus (Fig. 4D), as predicted from the absence of any other structural feature. This indicates that interaction of a 3' terminus with the 3' terminus binding pocket may be sufficient for stimulation of PriA ATP hydrolysis. However, the requirement of the minimum of 40 to 50 nucleotides for significant ATP hydrolysis suggests that PriA may need to interact not only with the 3'-end but also with other portions of DNA to be able to hydrolyze ATP. The extended single-stranded DNA of 50 nucleotides is about 30 nm long, approximately the length of nine turns of double helix. The footprinting and protection data indicate that the DNA helicase domain makes contact with about 20-base pair segments (two turns of double helix) of unreplicated duplex DNA of an arrested fork. We speculate that single-stranded DNA may need to adopt a compact structure, which enables its interaction not only with the 3' terminus binding pocket but also with the helicase domain.
On the other hand, A-fork [3'] can stimulate ATP hydrolysis even in the absence of 3'-recognition (Fig. 3, B-D), suggesting that DNA binding through the putative additional domain alone can also activate PriA ATPase. We propose that the N-terminal DNA binding domain (through either of the two recognition modules) facilitates the interaction of the DNA helicase domain with the effector DNA by first recognizing and binding to it. This bipartite interaction of effector DNA with PriA (through the N-terminal DNA binding domain and helicase domain) would cause conformational change of the helicase domain, which now relieves the inhibition and permits efficient hydrolysis of ATP. Because the isolated helicase domain completely lacks DNA binding activities (Ref. 23 and data not shown), the "activation" of DNA binding capacity of the helicase domain, caused by the initial interaction at the N-terminal DNA binding domain, would be crucial for ATPase activation of PriA.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S6 and Table S1.
1 Present address: Basic Analytical Chemistry Group, Institute of Life Sciences, AJINOMOTO CO., INC., Kawasaki, 210-8681, Japan.
2 To whom correspondence should be addressed. Tel.: 81-3-5685-2264; Fax: 81-3-5685-2932; E-mail: hmasai{at}rinshoken.or.jp.
3 The abbreviations used are: CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydropropanesulfonic acid; A-fork, arrested fork-like structure; AFM, A-fork [3'] mimic.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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