
f Co-expression and purification of the RadA recombinase with the RadB paralog from Haloferax volcanii yields heteromeric ring-like structures
- Authors: Bushra B. Patoli1,† , Jody A. Winter1,‡ , Atif A. Patoli1,† , Robin M. Delahay2 , Karen A. Bunting1,§
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1 1School of Biology, Queens Medical Centre, University of Nottingham, Nottingham, NG7 2UH, UK 2 2School of Medicine, University of Nottingham, Nottingham, NG7 2UH, UK † †Present address: Institute of Microbiology, University of Sindh, Jamshoro, Pakistan. ‡ ‡Present address: Nottingham Trent University, Nottingham, NG11 8NS, UK. § §Present address: Albumedix Ltd, Nottingham, NG7 1FD, UK.
- *Correspondence: Bushra B. Patoli [email protected]
- First Published Online: 26 October 2017, Microbiology 163: 1802-1811, doi: 10.1099/mic.0.000562
- Subject: Genomics and Systems Biology
- Received:
- Accepted:
- Cover date:




Co-expression and purification of the RadA recombinase with the RadB paralog from Haloferax volcanii yields heteromeric ring-like structures, Page 1 of 1
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The study of archaeal proteins and the processes to which they contribute poses particular challenges due to the often extreme environments in which they function. DNA recombination, replication and repair proteins of the halophilic euryarchaeon, Haloferax volcanii (Hvo) are of particular interest as they tend to resemble eukaryotic counterparts in both structure and activity, and genetic tools are available to facilitate their analysis. In the present study, we show using bioinformatics approaches that the Hvo RecA-like protein RadA is structurally similar to other recombinases although is distinguished by a unique acidic insertion loop. To facilitate expression of Hvo RadA a co-expression approach was used, providing its lone paralog, RadB, as a binding partner. At present, structural and biochemical characterization of Hvo RadA is lacking. Here, we describe for the first time co-expression of Hvo RadA with RadB and purification of these proteins as a complex under in vitro conditions. Purification procedures were performed under high salt concentration (>1 M sodium chloride) to maintain the solubility of the proteins. Quantitative densitometry analysis of the co-expressed and co-purified RadAB complex estimated the ratio of RadA to RadB to be 4 : 1, which suggests that the proteins interact with a specific stoichiometry. Based on a combination of analyses, including size exclusion chromatography, Western blot and electron microscopy observations, we suggest that RadA multimerizes into a ring-like structure in the absence of DNA and nucleoside co-factor.
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One supplementary table and three supplementary figures are available with the online version of this article.
- Keyword(s): Haloferax volcanii, homologous recombination, co-expression, RadA, halophilic protein purification
© 2017 The Authors
-
1. Kowalczykowski SC, Eggleston AK. Homologous pairing and DNA strand-exchange proteins. Annu Rev Biochem 1994; 63: 991– 1043 [CrossRef] [PubMed]
-
2. Story RM, Weber IT, Steitz TA. The structure of the E. coli recA protein monomer and polymer. Nature 1992; 355: 318– 325 [CrossRef] [PubMed]
-
3. Sandler SJ, Satin LH, Samra HS, Clark AJ. recA-like genes from three Archaean species with putative protein products similar to Rad51 and Dmc1 proteins of the yeast Saccharomyces cerevisiae. Nucleic Acids Res 1996; 24: 2125– 2132 [CrossRef] [PubMed]
-
4. Ogawa T, Shinohara A, Nabetani A, Ikeya T, Yu X et al. RecA-like recombination proteins in eukaryotes: functions and structures of RAD51 genes. Cold Spring Harb Symp Quant Biol 1993; 58: 567– 576 [CrossRef] [PubMed]
-
5. Shinohara A, Ogawa H, Ogawa T. Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell 1992; 69: 457– 470 [CrossRef] [PubMed]
-
6. Komori K, Miyata T, Daiyasu H, Toh H, Shinagawa H et al. Domain analysis of an archaeal RadA protein for the strand exchange activity. J Biol Chem 2000; 275: 33791– 33797 [CrossRef] [PubMed]
-
7. Ariza A, Richard DJ, White MF, Bond CS. Conformational flexibility revealed by the crystal structure of a crenarchaeal RadA. Nucleic Acids Res 2005; 33: 1465– 1473 [CrossRef] [PubMed]
-
8. Shin DS, Pellegrini L, Daniels DS, Yelent B, Craig L et al. Full-length archaeal Rad51 structure and mutants: mechanisms for RAD51 assembly and control by BRCA2. Embo J 2003; 22: 4566– 4576 [CrossRef] [PubMed]
-
9. Sung P, Klein H. Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat Rev Mol Cell Biol 2006; 7: 739– 750 [CrossRef] [PubMed]
-
10. Symington LS. Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiol Mol Biol Rev 2002; 66: 630– 670 [CrossRef] [PubMed]
-
11. Sandler SJ, Hugenholtz P, Schleper C, Delong EF, Pace NR et al. Diversity of radA genes from cultured and uncultured Archaea: comparative analysis of putative RadA proteins and their use as a phylogenetic marker. J Bacteriol 1999; 181: 907– 915 [PubMed]
-
12. Komori K, Miyata T, Diruggiero J, Holley-Shanks R, Hayashi I et al. Both RadA and RadB are involved in homologous recombination in Pyrococcus furiosus. J Biol Chem 2000; 275: 33782– 33790 [CrossRef] [PubMed]
-
13. Allers T, Ngo HP. Genetic analysis of homologous recombination in Archaea: Haloferax volcanii as a model organism. Biochem Soc Trans 2003; 31: 706– 710 [CrossRef] [PubMed]
-
14. Haldenby S, White MF, Allers T. RecA family proteins in Archaea: RadA and its cousins. Biochem Soc Trans 2009; 37: 102– 107 [CrossRef] [PubMed]
-
15. Guy CP, Haldenby S, Brindley A, Walsh DA, Briggs GS et al. Interactions of RadB, a DNA repair protein in archaea, with DNA and ATP. J Mol Biol 2006; 358: 46– 56 [CrossRef] [PubMed]
-
16. Allers T. Overexpression and purification of halophilic proteins in Haloferax volcanii. Bioeng Bugs 2010; 1: 290– 292 [CrossRef]
-
17. Allers T, Barak S, Liddell S, Wardell K, Mevarech M. Improved strains and plasmid vectors for conditional overexpression of His-tagged proteins in Haloferax volcanii. Appl Environ Microbiol 2010; 76: 1759– 1769 [CrossRef] [PubMed]
-
18. Wardell K, Haldenby S, Jones N, Liddell S, Ngo GHP et al. RadB acts in homologous recombination in the archaeon Haloferax volcanii, consistent with a role as recombination mediator. DNA Repair 2017; 55: 7– 16 [CrossRef] [PubMed]
-
19. Winter JA, Christofi P, Morroll S, Bunting KA. The crystal structure of Haloferax volcanii proliferating cell nuclear antigen reveals unique surface charge characteristics due to halophilic adaptation. BMC Struct Biol 2009; 9: 55 [CrossRef] [PubMed]
-
20. Winter JA, Patoli B, Bunting KA. DNA binding in high salt: analysing the salt dependence of replication protein A3 from the halophile Haloferax volcanii. Archaea 2012; 2012: 1– 12 [CrossRef] [PubMed]
-
21. Sheng D, Li M, Jiao J, Ni J, Shen Y. Co-expression with RadA and the characterization of stRad55B, a RadA paralog from the hyperthermophilic crenarchaea Sulfolobus tokodaii. Sci China C Life Sci 2008; 51: 60– 65 [CrossRef] [PubMed]
-
22. Sternberg N. Bacteriophage P1 cloning system for the isolation, amplification, and recovery of DNA fragments as large as 100 kilobase pairs. Proc Natl Acad Sci USA 1990; 87: 103– 107 [CrossRef] [PubMed]
-
23. Mullakhanbhai MF, Larsen H. Halobacterium volcanii spec. nov., a dead sea halobacterium with a moderate salt requirement. Arch Microbiol 1975; 104: 207– 214 [CrossRef] [PubMed]
-
24. Ziegler J, Vogt T, Miersch O, Strack D. Concentration of dilute protein solutions prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal Biochem 1997; 250: 257– 260 [CrossRef] [PubMed]
-
25. Janson G, Zhang C, Prado MG, Paiardini A. PyMod 2.0: improvements in protein sequence-structure analysis and homology modeling within PyMOL. Bioinformatics 2017; 33: 444– 446 [CrossRef] [PubMed]
-
26. Delano WL. The PyMol Molecular Graphics System Palo Alto, CA: DeLano Scientific LLC; 2008
-
27. Webb B, Sali A. Comparative protein structure modeling using MODELLER. Curr Protoc Bioinformatics 2016; 54: 5.6.1– 5.6.5 [Crossref]
-
28. Lanyi JK. Salt-dependent properties of proteins from extremely halophilic bacteria. Bacteriol Rev 1974; 38: 272– 290 [PubMed]
-
29. Chen LT, Ko TP, Chang YW, Lin KA, Wang AH et al. Structural and functional analyses of five conserved positively charged residues in the L1 and N-terminal DNA binding motifs of archaeal RADA protein. PLoS One 2007; 2: e858 [CrossRef] [PubMed]
-
30. Poidevin L, Macneill SA. Biochemical characterisation of LigN, an NAD+-dependent DNA ligase from the halophilic euryarchaeon Haloferax volcanii that displays maximal in vitro activity at high salt concentrations. BMC Mol Biol 2006; 7: 44 [CrossRef] [PubMed]
-
31. Gatzeva-Topalova PZ, May AP, Sousa MC. Crystal structure and mechanism of the Escherichia coli ArnA (PmrI) transformylase domain. An enzyme for lipid A modification with 4-amino-4-deoxy-L-arabinose and polymyxin resistance. Biochemistry 2005; 44: 5328– 5338 [CrossRef] [PubMed]
-
32. Breazeale SD, Ribeiro AA, Mcclerren AL, Raetz CR. A formyltransferase required for polymyxin resistance in Escherichia coli and the modification of lipid A with 4-Amino-4-deoxy-L-arabinose. Identification and function oF UDP-4-deoxy-4-formamido-L-arabinose. J Biol Chem 2005; 280: 14154– 14167 [CrossRef] [PubMed]
-
33. Yang S, Yu X, Seitz EM, Kowalczykowski SC, Egelman EH. Archaeal RadA protein binds DNA as both helical filaments and octameric rings. J Mol Biol 2001; 314: 1077– 1085 [CrossRef] [PubMed]
-
34. Dym O, Mevarech M, Sussman JL. Structural features that stabilize halophilic malate dehydrogenase from an archaebacterium. Science 1995; 267: 1344– 1346 [CrossRef] [PubMed]
-
35. Madern D, Ebel C, Zaccai G. Halophilic adaptation of enzymes. Extremophiles 2000; 4: 91– 98 [CrossRef] [PubMed]
-
36. Schlick T, Li B, Olson WK. The influence of salt on the structure and energetics of supercoiled DNA. Biophys J 1994; 67: 2146– 2166 [CrossRef] [PubMed]
-
37. Lee CD, Wang TF. The N-terminal domain of Escherichia coli RecA have multiple functions in promoting homologous recombination. J Biomed Sci 2009; 16: 37 [CrossRef] [PubMed]
-
38. Bergqvist S, Williams MA, O'Brien R, Ladbury JE. Halophilic adaptation of protein-DNA interactions. Biochem Soc Trans 2003; 31: 677– 680 [CrossRef] [PubMed]
-
39. Hartman AL, Norais C, Badger JH, Delmas S, Haldenby S et al. The complete genome sequence of Haloferax volcanii DS2, a model archaeon. PLoS One 2010; 5: e9605 [CrossRef] [PubMed]

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