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Analysis of interaction between DNA and Deinococcus radiodurans PprA protein by Atomic force microscopy
Biochimica et Biophysica Acta 1764 (2006) 20 – 23 http://www.elsevier.com/locate/bba
Analysis of interaction between DNA and Deinococcus radiodurans PprA protein by Atomic force microscopy Masahiro Murakami a,*, Issay Narumi b, Katsuya Satoh b, Akira Furukawa a, Isamu Hayata a a
Radiation Hazards Research Group, Research Center for Radiation Safety, National Institute of Radiological Sciences, 9-1, Anagawa-4-chome, Inage-ku, Chiba-shi 263-8555, Japan b Research Group for Gene Resources, Japan Atomic Energy Agency, 1233 Watanuki, Takasaki, Gunma 370-1292, Japan Received 19 January 2005; received in revised form 20 October 2005; accepted 20 October 2005 Available online 8 November 2005
Abstract A DNA repair-promoting protein, PprA, was isolated from a radiation resistant bacterium, Deinococcus radiodurans [I. Narumi, K. Sato, S. Cui, T. Funayama, S. Kitayama, H. Watanabe, PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation, Mol. Microbiol. 54 (2004) 278 – 285]. Despite several studies, however, the function of PprA is not still clear. We used atomic force microscopy (AFM) to elucidate the role of this protein in the DNA repair pathway. In the present study, interaction between the linear DNA and PprA protein was imaged and analyzed by AFM without any fixation or staining. Though both end-bound and internally bound PprA was observed, the affinity of the end-bound protein was greater considering the proportion of features of binding analyzed by AFM. In some conditions, looping forms of the DNA – PprA complex were observed. Gel filtration high performance liquid chromatography (HPLC) was also conducted to estimate the molecular weight of this protein. The result of the HPLC analysis suggested that PprA formed multimers in buffer solution without DNA. D 2005 Elsevier B.V. All rights reserved. Keywords: Atomic force microscopy; DNA repair; Radioresistant bacterium; Deinococcus radiodurans
1. Introduction The atomic force microscope is a tool used to visualize biological molecules at a resolution of nanometers [2]. For example, the interaction between DNA and DNA repair proteins such as the Ku heterodimer of vertebrates, DNAdependent protein kinase and Rad50/Mre11 was imaged and analyzed by AFM [3– 6]. The DNA-binding of the ataxiatelangiectasia gene product ATM protein was also visualized by AFM [7]. Deinococcus radiodurans shows the specific characteristic of being extremely radioresistant and the D37 dose (Dose of radiation required to reduce survival to 37%) of D. radiodurans is 6000 Gy of g-rays [8]. DNA polymerase I [9] and RecA [10] were required for the radioresistance of this bacterium. The presence of another repair system remains a possibility [11,12]. Recently, an unusual toroidal morphology
* Corresponding author. Tel.: +81 43 206 3077; fax: +81 43 255 6802. E-mail address: [email protected] (M. Murakami). 1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2005.10.017
that may contribute to D. radiodurans’s radioresistance was reported [13]. A unique radiation-inducible gene (designated pprA) responsible for loss of radiation resistance was identified by analyzing the DNA damage repair-deficient mutant (KH311) [1]. PprA (the gene product of pprA) is a novel DNA repair-promoting protein of D. radiodurans, whose molecular size is 32 kDa and the amino acid sequence deduced from pprA showed no homology to other known proteins [1]. This protein appears to be involved in DNA repair after binding with DNA as it promoted the ligation activities of NAD-dependent Escherichia coli DNA ligase and ATP-dependent T4 DNA ligase, and inhibited DNA degradation by E. coli exonuclease III [1]. The pprA disruptant strain was sensitive to g-rays [1]. PprA protein was induced when the bacterium was exposed to ionizing radiation [14,15]. The possibility that D. radiodurans has a non-homologous end joining (NHEJ) repair pathway has been discussed in several articles [1,16,17] and PprA is involved in NHEJ repair in D. radiodurans [1,16]. However, the function of PprA in the DNA repair system of D. radiodurans is not clear.
M. Murakami et al. / Biochimica et Biophysica Acta 1764 (2006) 20 – 23
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Fig. 1. Visualization of the interaction between the linear DNA and PprA protein by AFM. (A) Image of the DNA and PprA complex. End-bound and internal-bound PprA proteins were observed (arrow). Scale bar = 200 nm. (B) Three-dimensional representation of the DNA – PprA complex, Scale bar = 200 nm.
In the present study, the specific interaction between the linear DNA and the PprA protein was imaged and analyzed by AFM without any fixation or staining. 2. Materials and methods 2.1. Binding reactions of PprA and DNA PprA protein was isolated as reported previously [1]. Thirty-three nanograms of EcoRI-linearized and dephosphorylated plasmid DNA (pUC118 EcoRI/BAP, 3,162 bp, Takara Bio, Shiga, Japan) was mixed with 50 ng of PprA in a reaction buffer solution (10 mM Tris – HCl, pH 7.5, 10 mM MgCl2, 1 mM Dithiothreitol and 50 mM NaCl) and kept at 37 -C for 1 h. The DNA – protein mixture was then deposited onto freshly cleaved and discharged mica, rinsed with 100 Al of distilled water and blown dry with dry nitrogen.
2.2. Atomic force microscopy (AFM) DNA – protein complexes were visualized with an atomic force microscope (AFM, model SPI 3800N, Seiko Instruments Inc., Chiba, Japan) in the dynamic force mode (DFM) with a 20 Am scanner at room temperature [18 – 20]. The scan frequency was typically 2 Hz and all images contained 512 512 data points. Cantilevers for DFM-AFM (DF-40S, Seiko Instruments Inc., Chiba, Japan) were used for this experiment.
2.3. High performance liquid chromatography (HPLC) Fifty micrograms of PprA was diluted in buffer solution (10 mM Tris – HCl, pH7.5, 10 mM MgCl2 and 50 mM NaCl) and dialyzed (Slide-A-Lyzer MINI dialysis Units, MW 10,000, Pearce, USA) against 500 ml of the same buffer solution overnight. The PprA solution (100 Al) was applied to a gel filtration column (Ultraspherogel Sec 3000, Beckman, USA) equilibrated with 10 mM Tris – HCl, pH7.5, 10 mM MgCl2 and 50 mM NaCl, and eluted and analyzed by HPLC (System Gold, Beckman, USA). The flow rate was 1 ml/min and peaks of proteins were monitored by measuring absorbance at 280 nm. Aldolase (158 kDa), catalase (232 kDa), ferritin (440 kDa), thyroglobulin (669 kDa), riobonucease A (13.7 kDa), chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), albumin (67 kDa) and blue dextran 2000 (Gel filtration calibration kits, Amersham Pharmacia Biotech, USA) were utilized as marker. The V/V 0 value was plotted against the log of the molecular weights of proteins. The elution volume of proteins and void volume were V and V 0, respectively. The molecular weight of PprA was estimated from the V/V 0 value of the PprA using marker proteins as standards [21].
from the results of a gel electrophoretic mobility shift analysis of PprA [1], the binding of PprA to DNA was confirmed by AFM (Fig. 1). PprA proteins bound double-stranded DNA with free ends. Moreover, AFM imaging revealed that PprA was not localized only to the DNA-ends, i.e., internally bound PprA was also observed (Fig. 1). As shown in Table 1, the proportion of end-bound PprA and internally bound PprA was 41.9% and 58.1%, respectively. That PprA binds to DNA-ends as shown by AFM, should correlate with the results obtained by the gel electrophoretic mobility shift assay that show PprA binds to the linear form of DNA and open circular DNA but not closed circular DNA [1]. However, internally bound PprA was observed only by AFM. It might be important to consider the character of internal binding. When the molecular sizes of the linear DNA and the PprA protein observed by AFM were compared, PprA was found to be smaller. The molecular volume of PprA was estimated from the data obtained by AFM. After determining the height and radius, the molecular volume was calculated by treating the molecule as a segment of a sphere as presented in Eq. (1) [22,23]: Vm ¼ ðph=6Þ 3r2 þ h2 ; ð1Þ In Eq. (1), V m is the molecular volume and h and r are the height and radius of the protein, respectively. The molecular volume of PprA (DNA-unbound form) and the PprA –DNA complex estimated from the data obtained by the AFM analysis was 117 T 6 nm3 and 205 T 10 nm3, respectively. The length of the linear form of plasmid DNA with molecular size of 2,961 bp was estimated as about 1 Am by AFM and transmission electron microscopy [18]. Thus, PprA and the PprA –DNA complex have a smaller molecular volume than DNA. If PprA bound to DNA randomly, the proportion of internally bound protein should be larger than that of end bound protein. In the present study, however, the proportion of internally bound and Table 1 Binging of PprA protein to the linear form of DNA analyzed by AFM
3. Results and discussion Samples of the DNA – PprA mixture dried on the mica were directly visualized by atomic force microscopy. As expected
End bound Internally bound Total
Observed
Percent
126 175 301
41.9 58.1 100
22
M. Murakami et al. / Biochimica et Biophysica Acta 1764 (2006) 20 – 23
Fig. 2. AFM images of the DNA – PprA complex. (A) Looping of the linear DNA was observed after the reaction with PprA. Scale bar = 500 nm. (B) Other types of DNA-looping mediated by PprA. Scale bar = 500 nm.
end bound PprA was almost the same, revealing that the affinity of the DNA-end bound form is greater. When considering the function of PprA protein in DNA repair mechanisms, this DNA-binding property is important because end bound eukaryotic Ku proteins were observed to be involved in NHEJ DNA repair mechanisms by AFM [3,4]. It is also reported that Ku bound preferentially to DNA with free ends and Rad52 involved in homologous recombination binds preferentially to single-stranded DNA [24]. The molecular volume of the protein was calculated using Eq. (2) [22,23]: Vc ¼ ðM0 =N0 ÞðV1 þ dV2 Þ
ð2Þ
M 0 is the molecular weight, N 0 is Avogadro’s number, and V 1 and V 2 are the partial specific volumes of the individual protein (0.74 cm3 g1 and 1 cm3 g1 water, respectively). d is the extent of protein hydration (0.4 mol H2O/mol protein). Molecular weight of the PprA-monomer was estimated to be
32 kDa [1] and molecular volume (V c) calculated using Eq. (2) was 61 nm3. When this value (V c) is compared with the molecular volume (V m) measured by AFM, PprA protein (DNA-unbound form, V m = 117 T 6 nm3) may form dimer as the result of self-association. Molecular volume of PprA –DNA complex (V m = 205 T 10 nm3) was larger than that of PprA as the result of protein – DNA interaction. Regarding these results, PprA-dimer may bind to DNA ends and promote DNA repair. Another possibility that should be considered is that PprA is a monomer and molecular volume is overestimated by AFM, because the tip artifacts may affect the volume estimation of protein by AFM. Further analysis with more advanced techniques should be performed to determine the conformation of the PprA binding to DNA. In some conditions, looping structures of the DNA –PprA complex were observed (Fig. 2). The frequency of DNA looping and multi-DNA complex formation by PprA was
Fig. 3. (A) Chromatogram of gel filtration HPLC of PprA. The flow rate was 1 ml/min and the protein peaks were monitored by measuring absorbance at 280 nm. Arrows indicate the retention time of thyrogrobulin (669 kDa), aldrase (158 kDa) and ribonuclease A (13.7 kDa). (B) Molecular weight of PprA protein estimated by gel filtration HPLC. The elution volume and void volume were V and V0 , respectively and the void volume was estimated from the elution volume of blue dextran 2000. (1) riobonuclease A (13.7 kDa), (2) chymotrypsinogen A (25 kDa), (3) ovalbumin (43 kDa), (4) albumin (67 kDa), (5) aldolase (158 kDa), (6) catalase (232 kDa), (7) ferritin (440 kDa), (8) thyroglobulin (669 kDa).
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estimated and the frequencies of the PprA – DNA mixture and DNA alone were 0.26 and 0.12, respectively. It was reported that DNA-bound eukaryotic Ku heterodimer mediated the formation of DNA loops [3]. Considering these results, it seems that the function of PprA corresponds to that of Ku protein. Therefore, the DNA-looping by PprA may also indicate the ability to tether the DNA ends. This notion corresponds to the property of PprA that stimulates DNA ligation [1]. When considering the functions of PprA, it is important to analyze the conformation of the protein. The molecular weight of PprA in the buffer solution without DNA was estimated by gel filtration HPLC. A chromatogram of PprA showed that one major peak was detected in the higher molecular weight region (Fig. 3A). This result shows the possibility that PprA tends to form larger complexes without DNA under the present experimental conditions compared to the molecular weight of PprA-monomer which was estimated to be 32 kDa [1]. The molecular weight of PprA was estimated from the V/V 0 value of the PprA (Fig. 3B). The V/V 0 of PprA was 1.241 and the molecular weight estimated by gel filtration HPLC was 403.7 kDa. This result shows that PprA may form multimers (dodecamars) in the buffer solution without DNA, because the molecular weight of the PprA-monomer was estimated to be 32 kDa by SDS polyacrylamide gel electrophoresis [1]. Multimer formation of PprA may be related to DNA looping observed by AFM and DNA repair promoting activity of PprA. Ku proteins involved in NHEJ form heterodimer without DNA and form tetramer when bind to DNA in vitro [3]. Possibility of Ku self-association after DNA binding and a physical tethering of the broken DNA strands have been discussed [3]. Multimer formation suggested by HPLC analysis is quite different from the dimer formation of PprA observed by AFM analysis. Further analysis will be required to explain this difference and the functional importance of multimeric formation of PprA. Acknowledgement This work was supported by the Nuclear Crossover Research Project of the Ministry of Culture, Education, Sports, Science and Technology in Japan. References [1] I. Narumi, K. Sato, S. Cui, T. Funayama, S. Kitayama, H. Watanabe, PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation, Mol. Microbiol. 54 (2004) 278 – 285. [2] H.G. Hansma, J.H. Hoh, Biomolecular imaging with the atomic force microscope, Ann. Rev. Biophys. Biomol. Struct. 23 (1994) 115 – 139. [3] R.B. Cary, S.R. Peterson, J. Wang, D.G. Bear, E.M. Bradbury, D.J. Chen, DNA looping by Ku and the DNA-dependent protein kinase, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 4267 – 4272. [4] D. Pang, S. Yoo, W.S. Dynan, M. Jung, A. Dritschilo, Ku proteins join DNA fragments as shown by atomic force microscopy, Cancer Res. 57 (1997) 1412 – 1415.
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[5] M. Yaneva, T. Kowalewski, M.R. Lieber, Interaction of DNA-dependent protein kinase with DNA and with Ku: biochemical and atomic-force microscopy studies, EMBO J. 16 (1997) 5098 – 5112. [6] M. de Jager, J. van Noot, D.C. van Gent, C. Dekker, R. Kanaar, C. Wyman, Human Rad50/Mre11 is a flexible complex that can tether DNA ends, Mol. Cell 8 (2001) 1129 – 1135. [7] G.C.M. Smith, R.B. Cary, N.D. Lakin, B.C. Hann, S.H. Teo, D.J. Chen, S.P. Jackson, Purification and DNA binding properties of the ataxia – telangiectasia gene product ATM, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 11134 – 11139. [8] J.R. Battista, Against all odds: the survival strategies of Deinococcus radiodurans, Ann. Rev. Microbiol. 51 (1997) 203 – 224. [9] P.D. Gutman, P. Fuchs, L. Ouyang, K.W. Minton, Identification, sequencing, and targeted mutagenesis of a DNA polymerase gene required for the extreme radioresitance of Deinococcus radiodurans, J. Bacteriol. 175 (1993) 3581 – 3590. [10] K. Satoh, I. Narumi, M. Kikuchi, S. Kitayama, T. Yanagisawa, K. Yamamoto, H. Watanabe, Characterization of RecA424 and RecA670 proteins from Deinococcus radiodurans, J. Biochem. 131 (2002) 121 – 129. [11] J.R. Battista, A.M. Earl, M.J. Park, Why is Deinococcus radiodurans so resistant to ionizing radiation? Trends Mircrobiol. 7 (1999) 362 – 365. [12] J.R. Battista, Radiation resistance: the fragments that remain, Curr. Biol. 10 (2000) R204 – R205. [13] S. Levin-Zaidman, J. Englander, E. Shimoni, A.K. Sharma, K.W. Minton, A. Minsky, Ringlike structure of the Deinococcus radiodurans genome: a key to radioresistance? Science 299 (2003) 254 – 256. [14] Y. Liu, J. Zhou, M.V. Omelchenko, A.S. Beliaev, A. Venkateswaran, J. Stair, L. Wu, D.K. Thompson, D. Xu, I.B. Rogozin, E.K. Gaidamakova, M. Zhai, K.S. Makarova, E.V. Koonin, M.J. Daly, Transcriptome dynamics of Deinococcus radiodurans recovering from ionizing radiation, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 4191 – 4196. [15] Y. Hua, I. Narumi, G. Gao, B. Tian, K. Satoh, S. Kitayama, B. Shen, PprI: a general switch responsible for extreme radioresistance of Deinococcus radiodurans, Biochem. Biophys. Res. Commun. 306 (2003) 354 – 360. [16] I. Narumi, Unlocking radiation resistance mechanisms: still a long way to go, Trends Microbiol. 11 (2003) 422 – 425. [17] J. Englander, E. Klein, V. Brumfeld, A.K. Sharma, A.J. Doherty, A. Minsky, DNA toroids: framework for DNA repair in Deinococcus radiodurans and in germinating bacterial spores, J. Bacteriol. 186 (2004) 5973 – 5977. [18] M. Murakami, H. Hirokawa, I. Hayata, Analysis of radiation damage of DNA by atomic force microscopy in comparison with agarose gel electrophoresis studies, J. Biochem. Biophys. Methods 44 (2000) 31 – 40. [19] M. Murakami, M. Minamihisamatsu, K. Sato, I. Hayata, Structural analysis of heavy ion radiation-induced chromosome aberrations by atomic force microscopy, J. Biochem. Biophys. Methods 48 (2001) 293 – 301. [20] M. Murakami, R. Kanda, M. Minamihisamatsu, I. Hayata, Characterization of ionizing radiation-induced ring chromosomes by atomic force microscopy, Anal. Biochem. 334 (2004) 251 – 256. [21] J.R. Whitaker, Determination of molecular weights of proteins by gel filtration on Sephadex, Anal. Chem. 35 (1963) 1950 – 1953. [22] S.W. Schneider, J. La¨rmer, R.M. Henderson, H. Oberleithner, Molecular weights of individual proteins correlate with molecular volumes measured by atomic force microscopy, Pflu¨gers Arch. 435 (1998) 362 – 367. [23] L.I. Pietrasanta, D. Thrower, W. Hsieh, S. Rao, O. Stemmann, J. Lechner, J. Carbon, H. Hansma, Probing the Saccharomyces cerevisiae centromeric DNA (CEN DNA)-binding factor 3 (CBF3) kinetochore complex by using atomic force microscopy, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 3757 – 3762. [24] D. Ristic, M. Modesti, R. Kanaar, C. Wyman, Rad52 and Ku bind to different DNA structures produced early in double-strand break repair, Nucleic Acids Res. 31 (2003) 5229 – 5237.
Analysis of interaction between DNA and Deinococcus radiodurans PprA protein by Atomic force microscopy Masahiro Murakami a,*, Issay Narumi b, Katsuya Satoh b, Akira Furukawa a, Isamu Hayata a a
Radiation Hazards Research Group, Research Center for Radiation Safety, National Institute of Radiological Sciences, 9-1, Anagawa-4-chome, Inage-ku, Chiba-shi 263-8555, Japan b Research Group for Gene Resources, Japan Atomic Energy Agency, 1233 Watanuki, Takasaki, Gunma 370-1292, Japan Received 19 January 2005; received in revised form 20 October 2005; accepted 20 October 2005 Available online 8 November 2005
Abstract A DNA repair-promoting protein, PprA, was isolated from a radiation resistant bacterium, Deinococcus radiodurans [I. Narumi, K. Sato, S. Cui, T. Funayama, S. Kitayama, H. Watanabe, PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation, Mol. Microbiol. 54 (2004) 278 – 285]. Despite several studies, however, the function of PprA is not still clear. We used atomic force microscopy (AFM) to elucidate the role of this protein in the DNA repair pathway. In the present study, interaction between the linear DNA and PprA protein was imaged and analyzed by AFM without any fixation or staining. Though both end-bound and internally bound PprA was observed, the affinity of the end-bound protein was greater considering the proportion of features of binding analyzed by AFM. In some conditions, looping forms of the DNA – PprA complex were observed. Gel filtration high performance liquid chromatography (HPLC) was also conducted to estimate the molecular weight of this protein. The result of the HPLC analysis suggested that PprA formed multimers in buffer solution without DNA. D 2005 Elsevier B.V. All rights reserved. Keywords: Atomic force microscopy; DNA repair; Radioresistant bacterium; Deinococcus radiodurans
1. Introduction The atomic force microscope is a tool used to visualize biological molecules at a resolution of nanometers [2]. For example, the interaction between DNA and DNA repair proteins such as the Ku heterodimer of vertebrates, DNAdependent protein kinase and Rad50/Mre11 was imaged and analyzed by AFM [3– 6]. The DNA-binding of the ataxiatelangiectasia gene product ATM protein was also visualized by AFM [7]. Deinococcus radiodurans shows the specific characteristic of being extremely radioresistant and the D37 dose (Dose of radiation required to reduce survival to 37%) of D. radiodurans is 6000 Gy of g-rays [8]. DNA polymerase I [9] and RecA [10] were required for the radioresistance of this bacterium. The presence of another repair system remains a possibility [11,12]. Recently, an unusual toroidal morphology
* Corresponding author. Tel.: +81 43 206 3077; fax: +81 43 255 6802. E-mail address: [email protected] (M. Murakami). 1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2005.10.017
that may contribute to D. radiodurans’s radioresistance was reported [13]. A unique radiation-inducible gene (designated pprA) responsible for loss of radiation resistance was identified by analyzing the DNA damage repair-deficient mutant (KH311) [1]. PprA (the gene product of pprA) is a novel DNA repair-promoting protein of D. radiodurans, whose molecular size is 32 kDa and the amino acid sequence deduced from pprA showed no homology to other known proteins [1]. This protein appears to be involved in DNA repair after binding with DNA as it promoted the ligation activities of NAD-dependent Escherichia coli DNA ligase and ATP-dependent T4 DNA ligase, and inhibited DNA degradation by E. coli exonuclease III [1]. The pprA disruptant strain was sensitive to g-rays [1]. PprA protein was induced when the bacterium was exposed to ionizing radiation [14,15]. The possibility that D. radiodurans has a non-homologous end joining (NHEJ) repair pathway has been discussed in several articles [1,16,17] and PprA is involved in NHEJ repair in D. radiodurans [1,16]. However, the function of PprA in the DNA repair system of D. radiodurans is not clear.
M. Murakami et al. / Biochimica et Biophysica Acta 1764 (2006) 20 – 23
21
Fig. 1. Visualization of the interaction between the linear DNA and PprA protein by AFM. (A) Image of the DNA and PprA complex. End-bound and internal-bound PprA proteins were observed (arrow). Scale bar = 200 nm. (B) Three-dimensional representation of the DNA – PprA complex, Scale bar = 200 nm.
In the present study, the specific interaction between the linear DNA and the PprA protein was imaged and analyzed by AFM without any fixation or staining. 2. Materials and methods 2.1. Binding reactions of PprA and DNA PprA protein was isolated as reported previously [1]. Thirty-three nanograms of EcoRI-linearized and dephosphorylated plasmid DNA (pUC118 EcoRI/BAP, 3,162 bp, Takara Bio, Shiga, Japan) was mixed with 50 ng of PprA in a reaction buffer solution (10 mM Tris – HCl, pH 7.5, 10 mM MgCl2, 1 mM Dithiothreitol and 50 mM NaCl) and kept at 37 -C for 1 h. The DNA – protein mixture was then deposited onto freshly cleaved and discharged mica, rinsed with 100 Al of distilled water and blown dry with dry nitrogen.
2.2. Atomic force microscopy (AFM) DNA – protein complexes were visualized with an atomic force microscope (AFM, model SPI 3800N, Seiko Instruments Inc., Chiba, Japan) in the dynamic force mode (DFM) with a 20 Am scanner at room temperature [18 – 20]. The scan frequency was typically 2 Hz and all images contained 512 512 data points. Cantilevers for DFM-AFM (DF-40S, Seiko Instruments Inc., Chiba, Japan) were used for this experiment.
2.3. High performance liquid chromatography (HPLC) Fifty micrograms of PprA was diluted in buffer solution (10 mM Tris – HCl, pH7.5, 10 mM MgCl2 and 50 mM NaCl) and dialyzed (Slide-A-Lyzer MINI dialysis Units, MW 10,000, Pearce, USA) against 500 ml of the same buffer solution overnight. The PprA solution (100 Al) was applied to a gel filtration column (Ultraspherogel Sec 3000, Beckman, USA) equilibrated with 10 mM Tris – HCl, pH7.5, 10 mM MgCl2 and 50 mM NaCl, and eluted and analyzed by HPLC (System Gold, Beckman, USA). The flow rate was 1 ml/min and peaks of proteins were monitored by measuring absorbance at 280 nm. Aldolase (158 kDa), catalase (232 kDa), ferritin (440 kDa), thyroglobulin (669 kDa), riobonucease A (13.7 kDa), chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), albumin (67 kDa) and blue dextran 2000 (Gel filtration calibration kits, Amersham Pharmacia Biotech, USA) were utilized as marker. The V/V 0 value was plotted against the log of the molecular weights of proteins. The elution volume of proteins and void volume were V and V 0, respectively. The molecular weight of PprA was estimated from the V/V 0 value of the PprA using marker proteins as standards [21].
from the results of a gel electrophoretic mobility shift analysis of PprA [1], the binding of PprA to DNA was confirmed by AFM (Fig. 1). PprA proteins bound double-stranded DNA with free ends. Moreover, AFM imaging revealed that PprA was not localized only to the DNA-ends, i.e., internally bound PprA was also observed (Fig. 1). As shown in Table 1, the proportion of end-bound PprA and internally bound PprA was 41.9% and 58.1%, respectively. That PprA binds to DNA-ends as shown by AFM, should correlate with the results obtained by the gel electrophoretic mobility shift assay that show PprA binds to the linear form of DNA and open circular DNA but not closed circular DNA [1]. However, internally bound PprA was observed only by AFM. It might be important to consider the character of internal binding. When the molecular sizes of the linear DNA and the PprA protein observed by AFM were compared, PprA was found to be smaller. The molecular volume of PprA was estimated from the data obtained by AFM. After determining the height and radius, the molecular volume was calculated by treating the molecule as a segment of a sphere as presented in Eq. (1) [22,23]: Vm ¼ ðph=6Þ 3r2 þ h2 ; ð1Þ In Eq. (1), V m is the molecular volume and h and r are the height and radius of the protein, respectively. The molecular volume of PprA (DNA-unbound form) and the PprA –DNA complex estimated from the data obtained by the AFM analysis was 117 T 6 nm3 and 205 T 10 nm3, respectively. The length of the linear form of plasmid DNA with molecular size of 2,961 bp was estimated as about 1 Am by AFM and transmission electron microscopy [18]. Thus, PprA and the PprA –DNA complex have a smaller molecular volume than DNA. If PprA bound to DNA randomly, the proportion of internally bound protein should be larger than that of end bound protein. In the present study, however, the proportion of internally bound and Table 1 Binging of PprA protein to the linear form of DNA analyzed by AFM
3. Results and discussion Samples of the DNA – PprA mixture dried on the mica were directly visualized by atomic force microscopy. As expected
End bound Internally bound Total
Observed
Percent
126 175 301
41.9 58.1 100
22
M. Murakami et al. / Biochimica et Biophysica Acta 1764 (2006) 20 – 23
Fig. 2. AFM images of the DNA – PprA complex. (A) Looping of the linear DNA was observed after the reaction with PprA. Scale bar = 500 nm. (B) Other types of DNA-looping mediated by PprA. Scale bar = 500 nm.
end bound PprA was almost the same, revealing that the affinity of the DNA-end bound form is greater. When considering the function of PprA protein in DNA repair mechanisms, this DNA-binding property is important because end bound eukaryotic Ku proteins were observed to be involved in NHEJ DNA repair mechanisms by AFM [3,4]. It is also reported that Ku bound preferentially to DNA with free ends and Rad52 involved in homologous recombination binds preferentially to single-stranded DNA [24]. The molecular volume of the protein was calculated using Eq. (2) [22,23]: Vc ¼ ðM0 =N0 ÞðV1 þ dV2 Þ
ð2Þ
M 0 is the molecular weight, N 0 is Avogadro’s number, and V 1 and V 2 are the partial specific volumes of the individual protein (0.74 cm3 g1 and 1 cm3 g1 water, respectively). d is the extent of protein hydration (0.4 mol H2O/mol protein). Molecular weight of the PprA-monomer was estimated to be
32 kDa [1] and molecular volume (V c) calculated using Eq. (2) was 61 nm3. When this value (V c) is compared with the molecular volume (V m) measured by AFM, PprA protein (DNA-unbound form, V m = 117 T 6 nm3) may form dimer as the result of self-association. Molecular volume of PprA –DNA complex (V m = 205 T 10 nm3) was larger than that of PprA as the result of protein – DNA interaction. Regarding these results, PprA-dimer may bind to DNA ends and promote DNA repair. Another possibility that should be considered is that PprA is a monomer and molecular volume is overestimated by AFM, because the tip artifacts may affect the volume estimation of protein by AFM. Further analysis with more advanced techniques should be performed to determine the conformation of the PprA binding to DNA. In some conditions, looping structures of the DNA –PprA complex were observed (Fig. 2). The frequency of DNA looping and multi-DNA complex formation by PprA was
Fig. 3. (A) Chromatogram of gel filtration HPLC of PprA. The flow rate was 1 ml/min and the protein peaks were monitored by measuring absorbance at 280 nm. Arrows indicate the retention time of thyrogrobulin (669 kDa), aldrase (158 kDa) and ribonuclease A (13.7 kDa). (B) Molecular weight of PprA protein estimated by gel filtration HPLC. The elution volume and void volume were V and V0 , respectively and the void volume was estimated from the elution volume of blue dextran 2000. (1) riobonuclease A (13.7 kDa), (2) chymotrypsinogen A (25 kDa), (3) ovalbumin (43 kDa), (4) albumin (67 kDa), (5) aldolase (158 kDa), (6) catalase (232 kDa), (7) ferritin (440 kDa), (8) thyroglobulin (669 kDa).
M. Murakami et al. / Biochimica et Biophysica Acta 1764 (2006) 20 – 23
estimated and the frequencies of the PprA – DNA mixture and DNA alone were 0.26 and 0.12, respectively. It was reported that DNA-bound eukaryotic Ku heterodimer mediated the formation of DNA loops [3]. Considering these results, it seems that the function of PprA corresponds to that of Ku protein. Therefore, the DNA-looping by PprA may also indicate the ability to tether the DNA ends. This notion corresponds to the property of PprA that stimulates DNA ligation [1]. When considering the functions of PprA, it is important to analyze the conformation of the protein. The molecular weight of PprA in the buffer solution without DNA was estimated by gel filtration HPLC. A chromatogram of PprA showed that one major peak was detected in the higher molecular weight region (Fig. 3A). This result shows the possibility that PprA tends to form larger complexes without DNA under the present experimental conditions compared to the molecular weight of PprA-monomer which was estimated to be 32 kDa [1]. The molecular weight of PprA was estimated from the V/V 0 value of the PprA (Fig. 3B). The V/V 0 of PprA was 1.241 and the molecular weight estimated by gel filtration HPLC was 403.7 kDa. This result shows that PprA may form multimers (dodecamars) in the buffer solution without DNA, because the molecular weight of the PprA-monomer was estimated to be 32 kDa by SDS polyacrylamide gel electrophoresis [1]. Multimer formation of PprA may be related to DNA looping observed by AFM and DNA repair promoting activity of PprA. Ku proteins involved in NHEJ form heterodimer without DNA and form tetramer when bind to DNA in vitro [3]. Possibility of Ku self-association after DNA binding and a physical tethering of the broken DNA strands have been discussed [3]. Multimer formation suggested by HPLC analysis is quite different from the dimer formation of PprA observed by AFM analysis. Further analysis will be required to explain this difference and the functional importance of multimeric formation of PprA. Acknowledgement This work was supported by the Nuclear Crossover Research Project of the Ministry of Culture, Education, Sports, Science and Technology in Japan. References [1] I. Narumi, K. Sato, S. Cui, T. Funayama, S. Kitayama, H. Watanabe, PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation, Mol. Microbiol. 54 (2004) 278 – 285. [2] H.G. Hansma, J.H. Hoh, Biomolecular imaging with the atomic force microscope, Ann. Rev. Biophys. Biomol. Struct. 23 (1994) 115 – 139. [3] R.B. Cary, S.R. Peterson, J. Wang, D.G. Bear, E.M. Bradbury, D.J. Chen, DNA looping by Ku and the DNA-dependent protein kinase, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 4267 – 4272. [4] D. Pang, S. Yoo, W.S. Dynan, M. Jung, A. Dritschilo, Ku proteins join DNA fragments as shown by atomic force microscopy, Cancer Res. 57 (1997) 1412 – 1415.
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