Biochemical Characterization of a Mycobacteriophage Derived DnaB Ortholog Reveals New Insight into the Evolutionary Origin of DnaB Helicases

RESEARCH ARTICLE Biochemical Characterization of a Mycobacteriophage Derived DnaB Ortholog Reveals New Insight into the Evolutionary Origin of DnaB Helicases Priyanka Bhowmik, Sujoy K. Das Gupta* Department of Microbiology, Bose Institute, P1/12 C.I.T. Scheme VIIM, Kolkata 700054, West Bengal, India * Abstract OPEN ACCESS Citation: Bhowmik P, Das Gupta SK (2015) Biochemical Characterization of a Mycobacteriophage Derived DnaB Ortholog Reveals New Insight into the Evolutionary Origin of DnaB Helicases. PLoS ONE 10(8): e0134762. doi:10.1371/ journal.pone.0134762 Editor: Maria Sola, Molecular Biology Institute of Barcelona, CSIC, SPAIN Received: March 4, 2015 Accepted: July 13, 2015 Published: August 3, 2015 Copyright: © 2015 Bhowmik, Das Gupta. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The authors received no specific funding for this work. Competing Interests: The authors have declared that no competing interests exist. The bacterial replicative helicases known as DnaB are considered to be members of the RecA superfamily. All members of this superfamily, including DnaB, have a conserved Cterminal domain, known as the RecA core. We unearthed a series of mycobacteriophage encoded proteins in which the RecA core domain alone was present. These proteins were phylogenetically related to each other and formed a distinct clade within the RecA superfamily. A mycobacteriophage encoded protein, Wildcat Gp80 that roots deep in the DnaB family, was found to possess a core domain having significant sequence homology (Expect value < 10-5) with members of this novel cluster. This indicated that Wildcat Gp80, and by extrapolation, other members of the DnaB helicase family, may have evolved from a single domain RecA core polypeptide belonging to this novel group. Biochemical investigations confirmed that Wildcat Gp80 was a helicase. Surprisingly, our investigations also revealed that a thioredoxin tagged truncated version of the protein in which the N-terminal sequences were removed was fully capable of supporting helicase activity, although its ATP dependence properties were different. DnaB helicase activity is thus, primarily a function of the RecA core although additional N-terminal sequences may be necessary for fine tuning its activity and stability. Based on sequence comparison and biochemical studies we propose that DnaB helicases may have evolved from single domain RecA core proteins having helicase activities of their own, through the incorporation of additional N-terminal sequences. Introduction Mycobacteriophages infect and grow in mycobacteria [1]. Several members of the genus Mycobacterium such as M. tuberculosis are pathogenic [2]. A major reason why mycobacteriophages are of interest is because they can be used as tools to study the molecular genetics of mycobacteria [3, 4]. However, in more recent times, there has been a paradigm shift. Mycobacteriophages by themselves are becoming interesting systems to study because of their diversity [5]. PLOS ONE | DOI:10.1371/journal.pone.0134762 August 3, 2015 1 / 26 Helicase Activity of Mycobacteriophage Wildcat Gp80 With the availability of genome sequences corresponding to a large number of such phages, several comparative genomic studies have been done [6]. These studies have revealed many interesting features about mycobacteriophage genomes and proteomes [5, 7]. It is intriguing that nearly 50% or more proteins encoded by mycobacteriophages have no database matches. Hence, a huge pool of proteins with no known function exists in the mycobacteriophage metaproteome. By characterizing these proteins using either in silico or experimental methods or both, it should be possible to discover proteins having novel functions in the near future. The other interesting aspect about mycobacteriophages is genetic mosaicism which is usually caused by illegitimate recombination events [8]. Such events can bring together unrelated DNA segments resulting in the rapid evolution of proteins through, what is sometimes referred to as, domain-lego mechanism [9–11]. Thus, mycobacteriophage derived genes can also serve as useful models to examine how protein functions evolve. Our research is targeted towards characterizing various mycobacteriophage derived proteins, with the hope of discovering at least a few having novel functions. This endeavour led us to the characterization of a protein (D29Gp65) encoded by gene 65 of mycobacteriophage D29, the product of which turned out to be a novel structure specific nuclease [12]. D29Gp65 and its orthologs belong to a large family of proteins known as the RecA superfamily, named after RecA [13], the major recombinase found in Eubacteria, counterparts of which are also found in the Eukarya (Rad51 and Dmc1) and Archaea (RadA, RadB and aRadC) [14, 15]. All members of the superfamily have a conserved protein fold known as RecA fold which is capable of hydrolyzing nucleotide triphosphates (NTPs) [16]. The energy generated in the process is utilized by the members of this family to execute their respective functions, most of which are linked to DNA transaction processes, such as recombination (RecA) [13], DNA repair (RadA/ sms) [17] and unwinding (DnaB) [18]. The domain spanning the RecA fold alone is referred to in this study as the RecA core. Most of the proteins in this family have domains in addition to the RecA core which are apparently necessary to support their specific functions. The notable exceptions are the archaeal proteins RadB and aRadC, and also KaiC, the circadian rhythm regulating protein of cyanobacteria [19], all of which have only the RecA core domain and nothing else [15]. D29Gp65 also belongs to the same category having just the single domain corresponding to the RecA core. The observation that a single domain only protein like D29Gp65 has a specific biochemical function was intriguing. D29Gp65 shares significant homology with another mycobacteriophage encoded protein, Wildcat Gp80 (WCGp80), the subject matter of this study, that was predicted to be a DnaB type helicase. The primary function of helicases is to act as motor proteins to unwind double- stranded DNA, RNA and DNA-RNA hybrids [20]. The energy required for this purpose is derived from the hydrolysis of NTP. The helicases can be classified into six super-families of which DnaB which is the replicative helicase in Eubacteria, belongs to Superfamily 4 (SF4) [20]. DnaB helicases are characteristically hexameric in structure and unwind DNA in an unidirectional manner in the 5'-3' direction [21]. The sequence similarity of the core of WCGp80 with D29Gp65, suggests that it, and by extension all members of the DnaB helicase fa (...truncated)