Communicated by: Hisao Masai
Crystal structure and DNA-binding mode of Klebsiella pneumoniae primosomal PriB protein
Article first published online: 3 SEP 2012
© 2012 The Authors Journal compilation © 2012 by the Molecular Biology Society of Japan/Wiley Publishing Ltd
Genes to Cells
Volume 17, Issue 10, pages 837–849, October 2012
How to Cite
Huang, Y.-H., Lo, Y.-H., Huang, W. and Huang, C.-Y. (2012), Crystal structure and DNA-binding mode of Klebsiella pneumoniae primosomal PriB protein. Genes to Cells, 17: 837–849. doi: 10.1111/gtc.12001
- Issue published online: 24 SEP 2012
- Article first published online: 3 SEP 2012
- Manuscript Accepted: 28 JUL 2012
- Manuscript Received: 23 FEB 2012
- National Research Program for Genome Medicine, Taiwan. Grant Number: 100-3112-B-040-001
PriB is a primosomal DNA replication protein required for the re-initiation of replication in bacteria. In this study, we investigated the gene expression of PriB in Klebsiella pneumoniae (KpPriB) and characterized the gene product through crystal structural and functional analyses. Quantitative polymerase chain reaction analysis (Q-PCR) indicated that the 104-aa priB was expressed in K. pneumoniae with a CT value of 22.4. The crystal structure of KpPriB (Protein Data Bank entry: 4APV) determined at a resolution of 2.1 Å was similar to that of Escherichia coli PriB (EcPriB). KpPriB formed a single complex with single-stranded DNA (ssDNA) of different lengths, suggesting a highly cooperative process. Structure-based mutational analysis revealed that substitution at K18, F42, R44, W47, K82, K84, or K89 but not R34 in KpPriB had a significant effect on both ssDNA and double-stranded DNA (dsDNA) binding. Based on these findings, the known ssDNA interaction sites of PriB were expanded to include R44 and F42, thus allowing nucleic acids to wrap around the whole PriB protein.
The initiation and re-initiation of chromosomal DNA replication in bacteria for genome duplication is a complex process that relies on divergent multiprotein assembly for entry of the replicative DNA helicase (Benkovic et al. 2001). In Escherichia coli, replication starts with the loading of DnaB helicase at the unique oriC site – a process accomplished by DnaA (an oriC-specific recognition protein) and DnaC (a loader protein) (Mott & Berger 2007). However, the replication fork initiated at oriC can be arrested anywhere along the DNA, leading to failure of replication (Cox et al. 2000; McGlynn & Lloyd 2002; Masai et al. 2010). Thus, a reloading DnaB helicase for oriC-independent DNA replication is required for bacterial survival (Gabbai & Marians 2010). The replication restart primosome is a multiprotein complex that reactivates stalled DNA replication at the forks after DNA damage (Masai et al. 2010). To date, at least two DnaB helicase-recruiting pathways are known: the PriA-PriB-DnaT-DnaC-dependent reactions are the most effective on fork structures with no gaps in the leading strand, whereas the PriC-DnaC-dependent system preferentially uses fork structures with large gaps in the leading strand (Heller & Marians 2006a,b). In the PriA-directed pathway, PriB is the second protein to be assembled in the protein-DNA complex (Marians 2000), where it then stimulates PriA helicase activity (Cadman et al. 2005). PriB also stabilizes the binding of PriA to DNA hairpins, therefore facilitating the association of DnaT with the primosome (Liu et al. 1996). In an ATP- and DnaC-dependent manner, DnaB helicase is then loaded onto the complex and forms the complete primosome upon binding to DnaG primase (Bailey et al. 2007; Lo et al. 2009). Recruitment of DnaB helicase to the DNA results in reactivation of the repaired replication forks, allowing bidirectional DNA synthesis to resume.
Escherichia coli PriB exists as a homodimer, with each PriB monomer (104 aa) possessing an oligonucleotide/oligosaccharide-binding (OB)-fold structure (Liu et al. 2004; Lopper et al. 2004; Shioi et al. 2005). The single-stranded DNA (ssDNA)-binding site of PriB is located centrally in L45 loop within the dimer (Huang et al. 2006) and occupies 12 ± 1 nt of the total site size (Szymanski et al. 2010). The N-terminal (1–49 aa) region of PriB is crucial for dimerization, whereas the C-terminal (50–104 aa) region is crucial for ssDNA binding (Hsieh & Huang 2011). Although a dimer, PriB has only one ssDNA-binding site (Huang et al. 2006; Szymanski et al. 2010). PriB shares structural similarity with the DNA-binding domain of E. coli ssDNA-binding protein (EcSSB) (Raghunathan et al. 2000), except for differences in their ssDNA-binding modes (Huang et al. 2006).
Recently, several small proteins possessing ssDNA-binding activity have been discovered (Aravind et al. 2003; Jelinska et al. 2005; Luo et al. 2007; Paytubi et al. 2012), including a shorter 55-aa Klebsiella pneumoniae PriB (KpPriBc) documented by the NCBI (Hsieh & Huang 2011). Some of these small DNA-binding proteins have similar properties. For instance, similar to KpPriBc, CC1 is a 6-kDa, monomeric, basic protein adopting an OB-fold-like structure (Luo et al. 2007). However, a recent study using sequence alignment indicated that KpPriBc, which is much shorter in length than the well-studied EcPriB, probably resulted from the use of the wrong ATG as the priB start codon (Berg & Lopper 2011). Although gel filtration and some ssDNA-binding analyses suggest that KpPriB (104-aa) is similar to EcPriB in structure and function (Berg & Lopper 2011), the gene expression of KpPriB has never been analyzed and identified. As many prokaryotic genomes do not contain a recognizable homologue of priB (Dong et al. 2010), the expression of KpPriB should be analyzed carefully. In this study, we identified the expression of the priB gene in K. pneumoniae and characterized the gene product by crystal structural and mutational analyses.
Expression of priB gene in K. pneumoniae
To date, gene expression of the “putative” 104-aa PriB in K. pneumoniae has never been analyzed and identified. To test whether the 104-aa PriB could be expressed in K. pneumoniae, total RNA was extracted and reverse transcribed and then analyzed by Q-PCR using specific primers. We found that the 104-aa PriB was expressed with a CT value of 22.4 (Fig. 1). This low CT value suggests that PriB is a nonhousekeeping gene. Results experimentally confirmed that the 104-aa PriB was actually expressed in K. pneumoniae. The original version of K. pneumoniae PriB, KPN_04595 (NCBI), probably resulted from the use of the wrong ATG as the priB start codon (residue Met50 in KpPriB).
KpPriB binds to ssDNA
Electrophoretic mobility shift assay (EMSA) is a popular and well-established approach in molecular biology that allows the detection of distinct complexes (Huang 2012). In this study, EMSA of the binding of KpPriB to dT20–dT60 with different protein concentrations revealed that KpPriB could not form a stable complex with dT20 during electrophoresis (Fig. 2). Because some smears were observed, it appears that KpPriB can interact with dT20. In contrast, the longer dT homopolymers (i.e., dT25, dT30, dT35, dT40, dT45, dT50, dT55, and dT60) bind to KpPriB to form a single complex. These interactions appear to be highly cooperative because only one complex of KpPriB molecules bound per ssDNA was visible; no other obvious and distinctive complex or intermediate form was detected. Our findings indicate that the length of ssDNA required to enable the formation of a stable complex with the KpPriB molecule(s) is approximately 25 nt, as determined using EMSA.
ssDNA-binding ability of KpPriB
To compare the binding ability of KpPriB to ssDNA of different lengths, the midpoint values for input ssDNA binding, calculated from the titration curves of EMSA and referred to as [Protein]50, were quantified. They are summarized in Table 1. In general, the binding ability of KpPriB to ssDNA increased with length. However, the ability of KpPriB to bind ssDNA does not significantly change when the length of the dT homopolymer is furthermore increased to 50 nt.
|ssDNA substrate||[KpPriB]50 (μm)|
|dT25||18 ± 2|
|dT30||9 ± 2|
|dT35||8 ± 1|
|dT40||6 ± 1|
|dT45||6 ± 0.8|
|dT50||4 ± 0.5|
|dT55||4 ± 0.5|
|dT60||4 ± 0.5|
|The 15-mer ssDNA||ND|
|The 30-mer ssDNA||12 ± 1|
|The 45-mer ssDNA||9 ± 1|
|dA30||16 ± 2|
There is a conflicting report on the binding abilities of KpPriB protein. The concentration of KpPriB required for 50% of the 15–45-mer fluorescein-labeled ssDNA to be bound, determined using fluorescence polarization spectroscopy, is 45–62 nm (Berg & Lopper 2011). In addition, we also noted that the binding ability of KpPriB to a longer 45-base ssDNA is lower than that to a shorter 30-base ssDNA (Berg & Lopper 2011). To compare their results, we also studied the DNA binding by KpPriB under the same conditions using EMSA (Fig. 3). As expected, KpPriB could not form a stable complex with the 15-mer ssDNA substrate, and the longer 30-mer and 45-mer ssDNA substrates can bind to KpPriB to form a single complex, as in the case using the dT homopolymers (Fig. 2). However, the [KpPriB]50 for the 30-mer and 45-mer ssDNA substrates are 9–12 μm, a significantly lower value (~ 2–3 orders of magnitude) than that reported previously using fluorescence polarization spectroscopy (Berg & Lopper 2011). The lower values from EMSA are also found in other protein-DNA complexes (Wang et al. 2003) and probably due to the dissociation of the DNA-protein complexes during gel electrophoresis.
Base preference for ssDNA binding of KpPriB
We used dT30 (Fig. 2C) and dA30 (Fig. 2J) to test the base preference for KpPriB binding to purine and pyrimidine (Table 1). Similar to EcPriB (Liu et al. 2004) and KpPriBc (Hsieh & Huang 2011), KpPriB showed a preference for dT30 than dA30, indicating that KpPriB preferentially binds to pyrimidine than purine. However, the in vitro base preference of PriB, as well as other SSBs, is still unknown (Lohman & Ferrari 1994).
Crystal structure of KpPriB
KpPriB was crystallized and its structure was determined at a resolution of 2.1 Å (Table 2). The cell unit contains only one monomer of KpPriB, but its oligomerization state in solution is dimeric (Berg & Lopper 2011). The KpPriB monomer has an OB-fold domain, similar to EcPriB and Neisseria gonorrhoeae PriB (NgPriB) (Fig. 4). The majority of the electron density for KpPriB is of good quality, but a discontinuity is observed for residues 84–86 (K84, N85, and G86), and these three residues are absent from the model, suggesting that this region is highly dynamic.
|Cell dimension (Å)||a = 64.8|
|b = 36.9 β = 113.8|
|c = 39.5|
|Completeness (%)||95.6 (99.8)a|
|Rsymb (%)||6.4 (25.9)|
|Bond lengths (Å)||0.007|
|Bond angles (°)||1.001|
|In preferred regions||91 (96.8%)|
|In allowed regions||3 (3.2%)|
ssDNA-binding mode of KpPriB
Previously, we described the crystal structure of EcPriB in complex with ssDNA dT15; a single dT15 periodically interacts with two OB-fold from 2 symmetrically related PriB dimers in the crystal (Huang et al. 2006). Although the DNA-binding site is known to be located centrally in loop L45 of EcPriB, the length of DNA in the cocrystal structure is not long enough to wrap the nucleic acid around the protein dimer. Thus, the ssDNA-binding mode of PriB is still not completely clear. Based on the structure of the EcPriB-DNA complex, we manually superimposed the location of ssDNA with the structure of KpPriB (Fig. 5A). ssDNA-binding sites R13, K18, W47, K82, K84, and K89 that were revealed by EcPriB-DNA complex and functional analyses were well conserved in KpPriB (Fig. 5B). In order for nucleic acid to wrap around the whole KpPriB protein, there are two significant grooves that may serve as potential ssDNA-binding pocket on the protein surface (Fig. 5C): one is mediated by F42-R44 (Model I) and the other involved R34-R44 (Model II).
To determine which of the two models is used for KpPriB binding to ssDNA, alanine substitution mutants (i.e. K18A, R34A, F42A, R44A, W47A, K82A, K84A, and K89A) and the double mutant W47A/K82A were constructed and analyzed by EMSA (Fig. 6). The [Protein]50 values for the binding of these KpPriB variants to dT30 are summarized in Table 3. F42A has a [KpPriB]50 value that was 2.3-fold higher than that of wild-type KpPriB, whereas substitution at R34 had very little effect (~1.0-fold) on ssDNA binding compared with wild-type KpPriB. Results suggest that Model I is most likely used as the ssDNA-binding mode of KpPriB (Fig. 5C). The DNA interaction sites of PriB (i.e., K84, R13, K82, K89, W47, and K18) that were previously investigated using the crystal structure of EcPriB-ssDNA complex has been expanded to include R44 and F42 to allow for nucleic acid to fully wrap around the whole KpPriB protein.
|KpPriB variants||[KpPriB]50 (μm)||Fold|
|Wild type||9 ± 2||1.0|
|K18A||10 ± 2||1.1|
|R34A||9 ± 2||1.0|
|F42A||21 ± 3||2.3|
|R44A||21 ± 4||2.3|
|W47A||17 ± 3||1.9|
|K82A||35 ± 5||3.9|
|K84A||32 ± 4||3.6|
|K89A||35 ± 6||3.9|
|R34A, R44A||19 ± 3||2.1|
To strengthen the conclusion that Model I is the DNA-binding mode of PriB, two mutant proteins containing the double mutations, F42A/R44A and R34A/R44A, were constructed and analyzed by EMSA (Fig. 6J,K). F42A/R44A has a [KpPriB]50 value that was significantly higher than that of either F42A or R44A, whereas R34/R44A had very little effect on ssDNA binding compared with R44A. These results may firmly rule out the role of R34 for ssDNA binding (Table 3). Furthermore, a quadruple mutant, F42A/R44A/W47A/K82A was also generated and no band shift of this quadruple mutant was observed, indicating a dramatically impaired ability for ssDNA binding of this mutant. Previous report has demonstrated that K82A/K84A/K89A in EcPriB plays a very important role in ssDNA binding (Huang et al. 2006). Taken together, these results showed that not only the highly electropositive region in L45 loop, the aromatic residues F42 and W47 are also very critical for ssDNA binding by PriB. However, it should be noted that the Model I is still not a fully convincing model at this time. The length of ssDNA used in this model is only 18 nt (15 nt in the complex structure of 2CCZ plus 3 nt manually added) and in fact, 18 nt long ssDNA did not bind to KpPriB (Fig. 2).
Double-stranded DNA (dsDNA) binding of KpPriB
Despite the fact that both PriB and SSB have a classical OB-fold ssDNA-binding surface, they bind ssDNA using different strategies. Unlike SSB (Raghunathan et al. 2000), PriB binds ssDNA with the highly electrostatic positive L45 loop surface (Huang et al. 2006). This feature leads us to assess whether KpPriB binds dsDNA. The 22 base pairs (bp) dsDNA substrate for EMSA was prepared by annealing two oligonucleotides, of which one DNA strand was radiolabeled. As expected, KpPriB can bind this dsDNA (Fig. 7A). However, unexpectedly, we found that the [KpPriB]50 for this dsDNA binding is 5 ± 0.5 μm, a value higher than that for ssDNA dT30 binding of KpPriB (9 ± 2 μm).
To investigate the contribution of individual amino acid residues to dsDNA binding, alanine substitution mutants were constructed and analyzed by EMSA (Fig. 7), similarly to our study on ssDNA binding (Fig. 6). For the first time, the dsDNA-binding mode of PriB was assessed. The [Protein]50 values for the binding of these KpPriB variants to dsDNA are summarized in Table 4. Like ssDNA binding, these mutant proteins F42, R44, W47, K82, K84, and K89 but not R34 of KpPriB had low [Protein]50 values (approximately 1.8- to- 3.8-folds) compared with wild-type KpPriB for dsDNA binding. In addition, the mutant proteins containing the double mutations W47A/K82A and F42A/R44A, and the quadruple mutations F42A/R44A/W47A/K82A, exhibited significantly impaired ability for dsDNA binding of KpPriB. Thus, results from these structure-based mutational analyses indicated that the amino acid residues important for ssDNA binding were also crucial for dsDNA binding in KpPriB (see 'Discussion').
|KpPriB variants||[KpPriB]50 (μm)||Fold|
|Wild type||5 ± 0.5||1.0|
|R34A||5 ± 0.5||1.0|
|F42A||9 ± 1||1.8|
|R44A||9 ± 1||1.8|
|W47A||18 ± 1||3.6|
|K82A||19 ± 2||3.8|
|K84A||18 ± 1||3.6|
|K89A||18 ± 1||3.6|
|R34A, R44A||10 ± 1||2.0|
|F42A, R44A, W47A, K82A||>>50||>>10|
In this study, we investigated the crystal structure and DNA-binding mode of KpPriB, in which several results extend the knowledge in the field of PriB family. We also confirmed that the 104-aa PriB was expressed in K. pneumoniae (Fig. 1), but not the 55-aa KpPriBc (KPN_04595) (Hsieh & Huang 2011), originally documented in NCBI. Although a recent study using sequence alignment suggests that KpPriBc, which is much shorter in length than the well-studied EcPriB, resulted from the use of the wrong ATG as the priB start codon (Berg & Lopper 2011), we believe that the expression of the 104-aa KpPriB still needs to be carefully analyzed. As PriB is such a small protein, we do not know whether some prokaryotic genomes without a recognizable homologue of priB resulted from the use of the wrong ATG as the priB start codon, such as in the case of K. pneumoniae. This problem could be addressed by investigation using RT-PCR, as demonstrated in this study.
Many SSB proteins bind to ssDNA with some degree of positive cooperativity (Lohman & Ferrari 1994). In this study, we found differing EMSA behaviors between PriB and SSB proteins. SSB proteins form multiple distinct complexes with ssDNA of different lengths (Olszewski et al. 2008, 2010; Huang et al. 2011; Jan et al. 2011; Huang & Huang 2012), whereas KpPriB binding to ssDNA dT20-dT60 forms a single complex only (Fig. 2). EMSA is a useful technology in molecular biology that allows the detection of distinct complexes (Huang 2012). These findings strongly suggest that KpPriB binds to ssDNA with higher cooperativity than SSB proteins. However, it should be noted that ssDNA-binding affinity of PriB is significantly lower (>2–3 orders of magnitude) than that of SSB proteins (Huang et al. 2011; Jan et al. 2011; Huang & Huang 2012). Interestingly, sequence comparisons and operon organization analyses have shown that PriB evolved from SSB via gene duplication with subsequent rapid sequence diversification (Ponomarev et al. 2003). These findings raises several questions as to why PriB has become a kind of SSB with lower ssDNA-binding ability and higher cooperativity, and how PriB participates in DNA replication differently from its ancestor. Previous investigations have demonstrated that aromatic stacking plays an important role in ssDNA binding of SSB (Raghunathan et al. 2000), whereas PriB binds to the phosphate backbone of ssDNA via its highly electropositive region in L45 loops of the OB-fold (Huang et al. 2006). SSB possesses conserved aromatic residues (W40, W54, and F60) in L45 loop of the OB-fold; in contrast, two of these residues (W40 and F60) are replaced with nonconserved amino acids in the PriB family. A possible explanation is that during evolution, the conserved aromatic and other residues in L45 loop of the OB-fold in SSB were changed into positively charged residues in PriB in order to more precisely fit the requirement for assembly of the replication re-initiation primosome at the stalled DNA forks.
Results from the mutational analysis (Fig. 6) and the crystal structural information (Fig. 4) demonstrated that Model I was most likely used for ssDNA binding of KpPriB (Fig. 5C). Thus, the ssDNA interaction sites of PriB that were previously investigated using the crystal structure of EcPriB-ssDNA complex has been expanded to include R44 and F42 in order to allow for nucleic acid to fully wrap around the whole KpPriB protein. Although PriB uses a different DNA-binding strategy compared with other SSBs, including human replication protein A (Bochkarev et al. 1997; Huang et al. 2006), all SSB proteins still follow a similar DNA-binding path (i.e., loop L45 to loop L23) of OB-fold proteins (Murzin 1993; Theobald et al. 2003).
There are conflicting reports on the binding ability of residue 34 in PriB proteins. Substitution of K34 in NgPriB with alanine, the position structurally corresponding to R34 in KpPriB, significantly reduces the protein's ssDNA-binding activity (Dong et al. 2010). Results indicate that K34 probably plays an important role in NgPriB, whereas R34 in KpPriB does not. In addition, the binding ability of NgPriB to a (longer) 45-base ssDNA is ~2-fold lower than that to a (shorter) 36-base ssDNA (Dong et al. 2010). In contrast, the binding ability of KpPriB to oligonucleotides generally increases with greater length (Table 1). As the mechanisms for participating in assembly of the replication re-initiation primosome should be distinct (Dong et al. 2010; Feng et al. 2011), NgPriB and KpPriB may bind DNA differently. The crystal structure of NgPriB complexed with DNA is highly needed in helping our understanding of the primosome assembly mechanism(s).
In this study, we also found that KpPriB can bind both ssDNA and dsDNA with comparable affinity (Tables 1 and 4). According to crystal structures of some dimeric proteins complexed with dsDNA found in Protein Data Bank (PDB), as well as results from mutational analysis (Fig. 7), we speculate that KpPriB binds dsDNA in two possible ways (Fig. 8). First, KpPriB may bind to dsDNA via the HU-binding mode (Swinger et al. 2003; Kamashev et al. 2008). HU, a dimeric nucleoid-associated protein, mainly uses the two β sheets to bind the dsDNA (Fig. 8A). According to the structure of HU-DNA complex, we manually superimpose the location of dsDNA with our KpPriB structure and find that this complex structure seems to match the residues important for dsDNA binding (Fig. 7 and Table 4). Secondly, KpPriB may bind dsDNA in a manner similar to bind ssDNA (Fig. 8B). The structure-based mutational analysis indicated that residues in KpPriB crucial for ssDNA binding were also crucial for dsDNA binding (Tables 3 and 4). As these residues responsible for ssDNA and dsDNA binding were almost overlapped, KpPriB may use a similar way to bind to the phosphate backbone of ssDNA and dsDNA via several positively charged residues. This may be a reason for the comparable binding affinities of KpPriB with ssDNA and dsDNA. This might also explain why SSB can bind dsDNA but with far less affinity than ssDNA (Meyer & Laine 1990): several important residues in L45 loop of the OB-fold of SSB are aromatic residues (W40, W54, and F60), not the positively charged residues like in PriB. Taken together, we believe that both these two possible ways to bind dsDNA for KpPriB, namely the HU binding mode or ssDNA-binding mode of KpPriB, cannot be ruled out at this time.
The binding site on PriB for ssDNA overlaps the binding sites for PriA and DnaT, suggesting a dynamic primosome assembly process in which ssDNA is handed off from one primosome protein to another as a repaired replication fork is reactivated (Lopper et al. 2007). However, the complexed structure (Huang et al. 2006) and the thermodynamic analysis (Szymanski et al. 2010) indicate that the PriB dimer behaves like a protein with half-site reactivity, where only one monomer of the dimer can engage in interactions with the DNA and the partner protein(s) (Szymanski et al. 2010). Thus, it still remains to be explored whether the binding site on PriB for ssDNA is necessary to overlap the binding sites for PriA and DnaT.
The activity mediated by the replication primosomal proteins, including PriB, to reinitiate replication after DNA damage is essential for bacterial survival. There are some conflicting reports on the proposed binding mode of PriB to DNA and its partner proteins (Liu et al. 2004; Cadman et al. 2005; Huang et al. 2006; Lopper et al. 2007; Dong et al. 2010). It is not known whether this disparity is because of inherent differences among the species, use of different assay methods, or the effect of different investigators. This study reports the structural and functional analysis of KpPriB and discusses why PriB evolved from SSB to become a new DNA-binding protein during evolution. The more complex structures of PriB are useful in helping our understanding of the primosome assembly mechanism(s).
All restriction enzymes and DNA-modifying enzymes were purchased from New England Biolabs (Ipswich, MA, USA) unless explicitly stated otherwise. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless explicitly stated otherwise. The E. coli strains TOP10F' (Invitrogen, USA) and BL21(DE3)pLysS (Novagen, UK) were used for genetic construction and protein expression, respectively.
RNA purification and cDNA synthesis
Klebsiella pneumoniae subsp. pneumoniae MGH 78578 was grown to an OD600 of 1.0 at 37 °C in Luria-Bertani medium. RNA of K. pneumoniae was extracted from 4 mL cell cultures using RNAprotect Bacteria Reagent and Qiagen RNeasy Mini Columns (Qiagen), according to the manufacturer's instructions. Total RNA was treated with DNaseI and reverse transcribed using high-capacity cDNA reverse transcription kit (PE Applied Biosystems) and random primers according to the manufacturer's instructions.
Quantitative real-time PCR
Quantitative real-time PCR (Q-PCR) analysis was carried out using PowerSYBR Green Master Mix on a 7900 Real-Time PCR system (PE Applied Biosystems) as recommended by the manufacturer. Briefly, cDNA (25.4 ng) and primers (300 nm) were used in the amplification reactions: one cycle of 50 °C for 2 min; one cycle of 95 °C for 10 min; 45 cycles of 95 °C for 15 s and 60 °C for 20 s; and one cycle of 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. Primer 1 (5′-CACTGCCAGTTCGTGCTTGA-3′) and Primer 2 (5′-ACTGTGAGTAATGGCCTGGTTCTC-3′) were used. Product formation was monitored by the increase in fluorescence from SYBR Green intercalation. The threshold cycle (CT) is the first cycle for which a statistically significance increase in the amount of product is detected. CT values are inversely proportional to the cDNA amount in the sample. The expression plasmid pET21b-KpPriB served as quantitative standard.
Cloning, protein expression, and purification
The gene encoding the putative KpPriB was PCR-amplified using cDNA from the reverse transcribed RNA as template. The forward (GAAGGGGCATATGCCCGTTATTATTAGCGGTCATGAG) and reverse primers (GGGCTCGAGGTCTCCAGAATCTATCAATTCAAT) were designed to introduce unique NdeI and XhoI restriction sites into KpPriB, thus allowing the insertion of the amplified genes into the pET21b vector (Novagen Inc., Madison, WI, USA). The recombinant KpPriB protein was expressed and purified using the protocol used for EcPriB (Liu et al. 2004). Briefly, E. coli cells were transformed with the expression vector and grown to OD600 of 0.9 at 37 °C in Luria-Bertani medium containing 250 μg/mL ampicillin. Over-expression of KpPriB construct was induced with 1 mm isopropyl thiogalactoside (IPTG) for 3 h at 37 °C. The cells overexpressing the protein were chilled on ice, harvested by centrifugation, resuspended in Buffer A (20 mm Tris-HCl, 5 mm imidazole, 0.5 m NaCl; pH 7.9) and disrupted by sonication with ice cooling between pulses. The KpPriB protein was then purified from the soluble supernatant by Ni2+-affinity chromatography (HiTrap HP; GE Healthcare Bio-Sciences, Piscataway, NJ, USA). Protein purity was greater than 97% as determined by Coomassie-stained SDS-PAGE.
Preparation of dsDNA substrates
The dsDNA substrate (22 bp) was prepared with a radiolabeled strand (3′-GGGCTTAAGCTCGAGCCATGGG-5′) and an unlabeled strand (5′-CCCGAATTCGAGCTCGGTACCC-3′) at a 1 : 1 concentration ratio. The dsDNA substrate was formed in 20 mm HEPES (pH 7.0) and 100 mm NaCl, by brief heating at 95 °C for 5 min and then followed by slow cooling to room temperature overnight.
Electrophoretic mobility shift assay
Electrophoretic mobility shift assay for KpPriB was carried out according to the protocol described for SSB proteins (Huang et al. 2011; Jan et al. 2011; Huang & Huang 2012). Briefly, [γ32P]ATP (6000 Ci/mmol; PerkinElmer Life Sciences) and T4 polynucleotide kinase (Promega, Madison, WI, USA) were used for radiolabeling of various lengths of ssDNA oligonucleotides. KpPriB (0, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.5, 25, and 50 μm) was incubated with 1.7 nm DNA, 20 mm Tris-HCl pH 8.0, and 100 mm NaCl to a total volume of 10 μL for 30 min at 25 °C. Aliquots (5 μL) were removed from each reaction solution and added to 2 μL of gel-loading solution (0.25% bromophenol blue and 40% sucrose). The resulting samples were resolved on a native 8% polyacrylamide gel at 4 °C in TBE buffer (89 mm Tris borate and 1 mm EDTA) for 1 h at 100 V and visualized by autoradiography. Complexed and free DNA bands were scanned and quantified. For dsDNA, KpPriB (0, 0.4, 0.8, 1.6, 3.2, 6.4, 12.5, 25, and 50 μm) was used, and a radiolabeled ssDNA is shown in left lane as a control.
Other DNA substrates and conditions were also tried for comparison (Berg & Lopper 2011). KpPriB (0, 0.8, 1.6, 3.2, 6.4, 12.5, 25, and 50 μm) was incubated with 1 nm DNA (15-mer: 5′-TAGCAATGTAATCGT-3′; 30-mer: 5′-GCGTGGGTAATTGTGCTTCAATGGACTGAC-3′; 45-mer: 5′-GCCGTGATCACCAATGCAGATTGACGAACCTTTGCTCCAGTAACC-3′), 20 mm Tris–HCl pH 8.0, 50 mm NaCl, 4% glycerol, 1 mm MgCl2, 1 mm β-mercaptoethanol, 0.1 mg/mL bovine serum albumin to a total volume of 10 μL for 30 min at 25 °C, and then assayed using the protocol described earlier.
KpPriB mutants were generated according to the QuikChange Site-Directed Mutagenesis kit protocol (Stratagene, LaJolla, CA, USA) using the primers and wild-type plasmid pET21b-KpPriB as template. The presence of the mutation was verified by DNA sequencing.
The DNA-binding ability ([Protein]50) for the protein was estimated from the protein concentration that binds 50% of the input DNA. Each [Protein]50 is calculated as the average of at least three measurements ± SD.
Before crystallization, KpPriB was concentrated to 10 mg/mL in 20 mm sodium citrate and 100 mm NaCl (pH 5). Crystals were grown at room temperature by hanging drop vapor diffusion in 50% ethanol and 10 mm sodium acetate. Data collection and refinement statistics for the crystal of KpPriB are shown in Table 2. Data were collected using an ADSC Quantum-315r CCD area detector at SPXF beamline BL13C1 at NSRRC (Taiwan, ROC). All data integration and scaling were carried out using HKL-2000 (Otwinowski & Minor 1997). There was only one KpPriB molecule per asymmetric unit. The crystal structure of KpPriB was solved at 2.1 Å resolution with the molecular replacement software AMoRe (Navaza 1994) using EcPriB (Liu et al. 2004) as model (1V1Q). After molecular replacement, model building was carried out using XtalView (McRee 1999). CNS was used for molecular dynamic refinement (Brünger et al. 1998). The final structure was refined to an R-factor of 0.205 and an Rfree of 0.274. Atomic coordinates and related structure factors have been deposited in the PDB with accession code 4APV.
We would like to thank two anonymous reviewers and the editor for their comments. We also thank Prof. Chwan-Deng Hsiao (IMB of Academia Sinica) for technological support of this work. Portions of this study were carried out at the National Synchrotron Radiation Research Center, a national user facility supported by the National Science Council of Taiwan, ROC. The Synchrotron Radiation Protein Crystallography Facility is supported by the National Core Facility Program for Biotechnology. This research was supported by a grant from the National Research Program for Genome Medicine, Taiwan (NSC 100-3112-B-040-001 to C.Y. Huang).
- 2003) The two faces of Alba: the evolutionary connection between proteins participating in chromatin structure and RNA metabolism. Genome Biol. 4, R64. , & (
- 2007) Structure of hexameric DnaB helicase and its complex with a domain of DnaG primase. Science 318, 459–463. , & (
- 2001) Replisome-mediated DNA replication. Annu. Rev. Biochem. 70, 181–208. , & (
- 2011) The priB Gene of Klebsiella pneumoniae encodes a 104-amino acid protein that is similar in structure and function to Escherichia coli PriB. PLoS One 6, e24494. & (
- 1997) Structure of the single-stranded-DNA-binding domain of replication protein A bound to DNA. Nature 385, 176–181. , , & (
- 1998) Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. 54, 905–921. , , , , , , , , , , , , & (
- 2005) PriB stimulates PriA helicase via an interaction with single-stranded DNA. J. Biol. Chem. 280, 39693–39700. , , , & (
- 2000) The importance of repairing stalled replication forks. Nature 404, 37–41. , , , , & (
- 2010) The crystal structure of Neisseria gonorrhoeae PriB reveals mechanistic differences among bacterial DNA replication restart pathways. Nucleic Acids Res. 38, 499–509. , , , & (
- 2011) A bacterial PriB with weak single-stranded DNA binding activity can stimulate the DNA unwinding activity of its cognate PriA helicase. BMC Microbiol. 11, 189. , , & (
- 2010) Recruitment to stalled replication forks of the PriA DNA helicase and replisome-loading activities is essential for survival. DNA Repair 9, 202–209. & (
- 2006a) Replication fork reactivation downstream of a blocked nascent leading strand. Nature 439, 557–562. & (
- 2006b) Replisome assembly and the direct restart of stalled replication forks. Nat. Rev. Mol. Cell Biol. 7, 932–943. & (
- 2011) Identification of a novel protein, PriB, in Klebsiella pneumoniae. Biochem. Biophys. Res. Commun. 404, 546–551. & (
- 2006) Complexed crystal structure of replication restart primosome protein PriB reveals a novel single-stranded DNA-binding mode. Nucleic Acids Res. 34, 3878–3886. , , , & (
- 2012) Determination of the binding site-size of the protein-DNA complex by use of the electrophoretic mobility shift assay. In: Stoichiometry and Research – The Importance of Quantity in Biomedicine (ed. A. Innocenti), pp. 235–242. Rijeka, Croatia: InTech Press. (
- 2012) Characterization of a single-stranded DNA-binding protein from Klebsiella pneumoniae: mutation at either Arg73 or Ser76 causes a less cooperative complex on DNA. Genes Cells 17, 146–157. & (
- 2011) Characterization of a single-stranded DNA binding protein from Salmonella enterica Serovar Typhimurium LT2 . Protein J. 30, 102–108. , & (
- 2011) Characterization of a single-stranded DNA-binding protein from Pseudomonas aeruginosa PAO1. Protein J. 30, 20–26. , & (
- 2005) Obligate heterodimerization of the archaeal Alba2 protein with Alba1 provides a mechanism for control of DNA packaging. Structure 13, 963–971. , , , , , , & (
- 2008) HU binds and folds single-stranded DNA. Nucleic Acids Res. 36, 1026–1036. , , , & (
- 1996) The ordered assembly of the phiX174-type primosome. III. PriB facilitates complex formation between PriA and DnaT. J. Biol. Chem. 271, 15656–15661. , & (
- 2004) Crystal structure of PriB, a primosomal DNA replication protein of Escherichia coli. J. Biol. Chem. 279, 50465–50471. , , , , , & (
- 2009) The crystal structure of a replicative hexameric helicase DnaC and its complex with single-stranded DNA. Nucleic Acids Res. 37, 804–814. , , , , & (
- 1994) Escherichia coli single-stranded DNA-binding protein: multiple DNA-binding modes and cooperativities. Annu. Rev. Biochem. 63, 527–570. & (
- 2007) A hand-off mechanism for primosome assembly in replication restart. Mol. Cell 26, 781–793. , , & (
- 2004) Crystal structure of PriB, a component of the Escherichia coli replication restart primosome. Structure 12, 1967–1975. , & (
- 2007) CC1, a novel crenarchaeal DNA binding protein. J. Bacteriol. 189, 403–409. , , , , & (
- 2000) PriA-directed replication fork restart in Escherichia coli. Trends Biochem. Sci. 25, 185–189. (
- 2010) Stalled replication forks: making ends meet for recognition and stabilization. BioEssays 32, 687–697. , & (
- 2002) Recombinational repair and restart of damaged replication forks. Nat. Rev. Mol. Cell Biol. 3, 859–870. & (
- 1999) XtalView/Xfit–A versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125, 156–165. (
- 1990) The single-stranded DNA-binding protein of Escherichia coli. Microbiol. Rev. 54, 342–380. & (
- 2007) DNA replication initiation: mechanisms and regulation in bacteria. Nat. Rev. Microbiol. 5, 343–354. & (
- 1993) OB(oligonucleotide/oligosaccharide binding)-fold: common structural and functional solution for non-homologous sequences. EMBO J. 12, 861–867. (
- 1994) AMoRe: an automated package for molecular replacement. Acta Crystallogr. 50, 157–163. (
- 2010) Characterization of exceptionally thermostable single-stranded DNA-binding proteins from Thermotoga maritima and Thermotoga neapolitana. BMC Microbiol. 10, 260. , , , , & (
- 2008) Two highly thermostable paralogous single-stranded DNA-binding proteins from Thermoanaerobacter tengcongensis. Arch. Microbiol. 190, 79–87. , & (
- 1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326. & (
- 2012) Displacement of the canonical single-stranded DNA-binding protein in the Thermoproteales. Proc. Natl Acad. Sci. USA 109, E398–E405. , , , , , , , & (
- 2003) Gene duplication with displacement and rearrangement: origin of the bacterial replication protein PriB from the single-stranded DNA-binding protein Ssb. J. Mol. Microbiol. Biotechnol. 5, 225–229. , , & (
- 2000) Structure of the DNA binding domain of E. coli SSB bound to ssDNA. Nat. Struct. Biol. 7, 648–652. , , & (
- 2005) Crystal structure of a biologically functional form of PriB from Escherichia coli reveals a potential single-stranded DNA-binding site. Biochem. Biophys. Res. Commun. 326, 766–776. , , , , , , & (
- 2003) Flexible DNA bending in HU-DNA cocrystal structures. EMBO J. 22, 3749–3760. , , & (
- 2010) Interactions of the Escherichia coli primosomal PriB protein with the single-stranded DNA. Stoichiometries, intrinsic affinities, cooperativities, and base specificities. J. Mol. Biol. 398, 8–25. , & (
- 2003) Nucleic acid recognition by OB-fold proteins. Annu. Rev. Biophys. Biomol. Struct. 32, 115–133. , & (
- 2003) Nucleic acid binding properties of the nucleic acid chaperone domain of hepatitis delta antigen. Nucleic Acids Res. 31, 6481–6492. , , , , , , & (