Downloaded from genome.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press Article The Bacterial Replicative Helicase DnaB Evolved from a RecA Duplication Detlef D. Leipe,1 L. Aravind,2,3 Nick V. Grishin,1,4 and Eugene V. Koonin1,5 1National Center for Biotechnology Information (NCBI), National Library of Medicine, National Institutes of Health, Bethesda Maryland 20894 USA; 2Department of Biology, Texas A&M University, College Station, Texas 70843 USA The RecA/Rad51/DCM1 family of ATP-dependent recombinases plays a crucial role in genetic recombination and double-stranded DNA break repair in Archaea, Bacteria, and Eukaryota. DnaB is the replication fork helicase in all Bacteria. We show here that DnaB shares significant sequence similarity with RecA and Rad51/DMC1 and two other related families of ATPases, Sms and KaiC. The conserved region spans the entire ATP- and DNA-binding domain that consists of about 250 amino acid residues and includes 7 distinct motifs. Comparison with the three-dimensional structure of Escherichia coli RecA and phage T7 DnaB (gp4) reveals that the area of sequence conservation includes the central parallel ␤-sheet and most of the connecting helices and loops as well as a smaller domain that consists of a amino-terminal helix and a carboxy-terminal ␤-meander. Additionally, we show that animals, plants, and the malarial Plasmodium but not Saccharomyces cerevisiae encode a previously undetected DnaB homolog that might function in the mitochondria. The DnaB homolog from Arabidopsis also contains a DnaG–primase domain and the DnaB homolog from the nematode seems to contain an inactivated version of the primase. This domain organization is reminiscent of bacteriophage primases–helicases and suggests that DnaB might have been horizontally introduced into the nuclear eukaryotic genome via a phage vector. We hypothesize that DnaB originated from a duplication of a RecA-like ancestor after the divergence of the bacteria from Archaea and eukaryotes, which indicates that the replication fork helicases in Bacteria and Archaea/Eukaryota have evolved independently. Genetic recombination is an essential process for both show a meiotic arrest phenotype, and it probably func- recombinational repair and sexual reproduction. In tions in the formation of synaptonemal complexes and Bacteria, the central role in recombination is played by also in double-strand break repair (Bishop et al. 1992; the RecA recombinase enzyme (Radding 1989; Kowal- Dresser et al. 1997; Yoshida et al. 1998). Thus, there is czykowski and Eggleston 1994; Seitz et al. 1998). RecA functional overlap between Rad51 and DMC1 (Shino- is a DNA-dependent ATPase that promotes homolo- hara et al. 1997) and Caenorhabditis elegans seems to gous pairing and strand exchange between different have only a single Rad51/DMC1 homolog (Takanami double-stranded (ds) DNA molecules and is therefore et al. 1998). A Rad51/DMC1 homolog (termed RadA) necessary for homologous recombination and DNA re- that catalyzes DNA pairing and strand exchange (Seitz pair (Kowalczykowski et al. 1994). The biochemical ac- et al. 1998) is also found in the Archaea (Sandler et al. tivities of RecA include the ability to form regular he- 1996). lical filaments, bind single-stranded (ss) and dsDNA, The RecA/RadA/DMC1 recombinases are closely and bind and hydrolyze nucleoside triphosphates related to three other groups of ATPases, namely bac- (Kowalczykowski et al. 1994). In addition to its direct terial Sms (also called RadA), bacterial DnaB, and ar- role in recombination, RecA functions as a cofactor in chaeal and bacterial KaiC. The Sms protein is a poorly the cleavage reaction for LexA, the repressor of the SOS characterized bacterial homolog of RecA in which the regulon (Little and Mount 1982; Witkin 1991). There RecA ATPase domain is fused to a Zn ribbon and a are two types of RecA-like proteins in many eukaryotes, predicted serine protease domain (Koonin et al. 1996; namely Rad51 and DMC1/Lim15. Rad51 is expressed Aravind et al. 1999) (hereafter we use the designation in both meiotic and mitotic cells and mainly partici- Sms to avoid confusion with the archaeal RadA). Esch- pates in recombinational repair of double-strand erichia coli sms mutants show increased sensitivity to X breaks (Shinohara et al. 1992; Doutriaux et al. 1998). rays, UV radiation, and methyl methanesulfonate, sug- DMC1 is expressed in meiotic cells, its null mutants gesting a role in repair for the Sms protein (Neuwald et al. 1992; Song and Sargentini 1996). 3Present address: National Center for Biotechnology Information, National The cyanobacterial KaiABC gene cluster consti- Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894 USA. tutes the circadian clock in the cyanobacterium Sy- 4Present address: Department of Biochemistry, University of Texas Southwest- nechococcus (Ishiura et al. 1998; Iwasaki et al. 1999). The ern Medical Center, Dallas, Texas 75235 USA. KaiC protein generates a circadian oscillation by nega- 5Corresponding author. E-MAIL [email protected]; FAX (301) 435-7794. tive feedback control on its own expression (Ishiura et 10:5–16 ©2000 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/99 $5.00; www.genome.org Genome Research 5 www.genome.org Downloaded from genome.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press Leipe et al. al. 1998). The Synechococcus KaiC protein is composed yeast (Game 1993), XRCC2 (Tambini et al. 1997), R5H2 of two RecA-like domains joined head to tail. Highly and R5H3 (Cartwright et al. 1998), and TRAD (Kawa- conserved homologs of KaiC are found in the cyano- bata and Sacki 1998) in mammals, and several other bacterium Synechocystis, the bacterium Thermotoga, distinct RecA homologs found in Archaea and some and in all Archaea but absent from other bacteria and bacteria (Aravind et al. 1999). Some of these orphan eukaryotes (Makarova et al. 1999). RecA homologs appear to contain an inactivated The DnaB helicase is a crucial protein in bacterial ATPase domain (Aravind et al. 1999). Additional do- DNA replication. It unwinds the DNA duplex ahead of mains associated with the RecA core include a modi- the replication fork and is also responsible for attract- fied amino-terminal helix–hairpin–helix (HhH) do- ing the DnaG primase to the replication fork (Tougu et main in the archaeoeukaryotic RadA/DMC1, a amino- al. 1994; Lu et al. 1996). The active form of the protein terminal zinc finger and a carboxy-terminal Lon-type is a hexamer of identical 52.3-kD subunits that can protease domain in Sms, and a GTPase in one of the form rings with threefold (C3) and sixfold (C6) sym- archaeal RecA homologs (Aravind et al. 1999). metry (Yu et al. 1996) and it has been hypothesized Here, using a combination of sequence database that the amino-terminal ATPase domains of two adja- searches, sequence alignments, phylogenetic analysis, cent protomers dimerizes to make the C6–C3 conver- and structural comparison, we show that (1) DnaB, sion (Fass et al. 1999). The crystal structure of the he- RecA, DMC1/RadA, Sms, and KaiC share significant se- licase domain of phage T7 helicase–primase (gp4) has quence similarity along a region of 250 amino acids recently been solved (Sawaya et al. 1999) and it has that includes both the ATP-binding domain and the been found that the structure of the T7 helicase do- DNA-binding site; (2) DnaB likely evolved from RecA main and its interactions with neighboring subunits in by a gene duplication event at the onset of the evolu- the crystal resemble those of the RecA and F1 ATPase tion of the Bacteria; (3) RecA and DnaB are likely to (Sawaya et al. 1999). In addition to the ATPase domain, perform their function by a similar mechanism of con- E. coli DnaB comprises a globular amino-terminal do- formational change; (4) eukaryotes encode diverged main (proteolytic fragment III) that is essential for in- homologs of DnaB, some of which also contain a teraction with other proteins involved in DNA replica- DnaG-type primase domain; these genes might have tion like DnaA, DnaC, and the DnaG primase (Na- been introduced into the eukaryotic genome by a hori- kayama et al. 1984; Biswas et al. 1994; Sutton et al. zontal transfer event involving a bacteriophage. We 1998). The domain consists of six ␣ helices (Weigelt et hypothesize that the common ancestor of the RecA/ al. 1998; Fass et al. 1999; Weigelt et al. 1999) that are DnaB superfamily functioned as a recombinase in the attached to the carboxy-terminal ATPase domain by a last common ancestor (LCA) of all extant cells and that flexible hinge (Miles et al. 1997). a RecA homolog (DnaB) was recruited for the helicase In addition to RecA, DMC1/Rad51/RadA, DnaB, function at the replication fork once DNA replication Sms, and KaiC, there is a large number of proteins with evolved in bacteria. This interpretation lends further more limited phylogenetic distribution that contain support to the hypothesis that the DNA replication the core RecA ATPase domain. These include, among machinery evolved independently in bacteria and ar- others, Rad51-interacting proteins Rad55 and Rad57 in chaea/eukaryotes (Leipe et al. 1999). Figure 1 (See pages 7–9.) Multiple alignment of the core domain of the RecA/DnaB superfamily of ATPases. From top to bottom (separated by horizontal lines) the alignment contains sequences from bacterial and chloroplast DnaB, DnaB proteins and primase– helicase proteins from bacteriophages and eukaryotes, bacterial Sms proteins, KaiC from Archaea and Bacteria, RecA recombinase from Bacteria and phage T4, and RadA and Rad51/DMC1 recombinases from Archaea and Eukaryota. The 80% consensus for these proteins is shown below the aligned sequences. Numbers indicate the distance to the amino-terminal methionine and the carboxyl terminus of each protein and residues omitted within the alignment.