The RepA Protein of Plasmid RSF1010 Is a Replicative DNA Helicase

Scherzinger, Eberhard; Ziegelin, Günter; Bárcena, Montserrat; Carazo, José Marı́a; Lurz, Rudi; Lanka, Erich

doi:10.1074/jbc.272.48.30228

Cited by 56 publications

(102 citation statements)

References 61 publications

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“…Before centrifugation, TrbB and HP0525 were incubated in buffer F for 10 min at 30°C. Fractions were tested for dATPase activity as published elsewhere (20,30). Aliquots were electrophoresed in SDS͞15% polyacrylamide gels.…”

Section: Discussionmentioning

confidence: 99%

Sequence-related protein export NTPases encoded by the conjugative transfer region of RP4 and by the cag pathogenicity island of Helicobacter pylori share similar hexameric ring structures

Krause

Bárcena

Pansegrau

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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RP4 TrbB, an essential component of the conjugative transfer apparatus of the broad-host-range plasmid RP4, is a member of the PulE protein superfamily involved in multicomponent machineries transporting macromolecules across the bacterial envelope. PulE-like proteins share several well conserved motifs, most notable a nucleoside triphosphate binding motif (P-loop). Helicobacter pylori HP0525 also belongs to the PulE superfamily and is encoded by the pathogenicity island cag , involved in the inflammatory response of infected gastric epithelial cells in mammals. The native molecular masses of TrbB and HP0525 as determined by gel filtration and glycerol gradient centrifugation suggested a homohexameric structure in the presence of ATP and Mg 2+ . In the absence of nucleotides and bivalent cations, TrbB behaved as a tetramer whereas the hexameric state of HP0525 remained unaffected. Electron microscopy and image processing demonstrated that TrbB and HP0525 form ring-shaped complexes (diameter: 12 nm) with a central region (diameter: 3 nm) of low electron density when incubated in the presence of ATP and Mg 2+ . However, the TrbB average image appeared to be more elliptical with strong twofold rotational symmetry whereas HP0525 complexes are regular hexagons. Six well defined triangle-shaped areas of high electron density were distinguishable in both cases. Covalent crosslinking of TrbB suggests that the hexameric ring is composed from a trimer of dimers, because only dimeric, tetrameric, and hexameric species were detectable. The toroidal structure of TrbB and HP0525 suggests that both proteins catalyze a repetitive process, most probably translocating a cognate substrate across the inner membrane.

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Section: Discussionmentioning

confidence: 99%

Sequence-related protein export NTPases encoded by the conjugative transfer region of RP4 and by the cag pathogenicity island of Helicobacter pylori share similar hexameric ring structures

Krause

Bárcena

Pansegrau

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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“…More generally, we note that the 34-kDa C-terminal helicase domain of E1 is similar in size to the smallest naturally occurring hexameric helicase identified to date, the RepA protein (30 kDa) from the autonomously replicating RSF1010 plasmid (80). Furthermore, enzymatically active C-terminal domains of similar size have been identified for both E. coli DnaB (81-83) and phage T7 gene 4 protein (84), which are hexameric helicases in the same F4 family as RepA (85).…”

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confidence: 96%

Characterization of Recombinant HPV6 and 11 E1 Helicases

White

Pelletier

Brault

et al. 2001

Journal of Biological Chemistry

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To better characterize the enzymatic activities required for human papillomavirus (HPV) DNA replication, the E1 helicases of HPV types 6 and 11 were produced using a baculovirus expression system. The purified wild type proteins and a version of HPV11 E1 lacking the N-terminal 71 amino acids, which was better expressed, were found to be hexameric over a wide range of concentrations and to have helicase and ATPase activities with relatively low values for K m (ATP) of 12 M for HPV6 E1 and 6 M for HPV11 E1. Interestingly, the value of K m (ATP) was increased 7-fold in the presence of the E2 transactivation domain. In turn, ATP was found to perturb the co-operative binding of E1 and E2 to DNA. Mutant and truncated versions of in vitro translated E1 were used to identify a minimal ATPase domain composed of the C-terminal 297 amino acids. This fragment was expressed, purified, and found to be fully active in ATP hydrolysis, single-stranded DNA binding, and unwinding assays, despite lacking the minimal origin-binding domain.The papillomaviruses (PVs) 1 are small, nonenveloped DNA viruses that infect and replicate in the cutaneous or mucosal epithelia of humans and other mammals. There are over 100 types of human papillomavirus (HPV), which cause conditions ranging from plantar warts (HPV1) and genital warts (HPV6 and -11) to cervical cancer (HPV16, -18, and -31). HPV6 and -11 are also responsible for laryngeal papillomatosis, a rare but very serious infection of the respiratory tract (1). Antiviral agents capable of specifically inhibiting PV replication could play an important role in the treatment of these diseases, but none exist at this time.Despite their host and tissue specificity, all of the PV types share a common genomic organization. The closed, circular genome of ϳ8000 base pairs codes for only 10 proteins: eight early proteins termed E1-E8 and two late proteins, L1 and L2, that make up the viral coat (2). E1 and E2 are the only viral proteins required for HPV DNA replication (3, 4). E2 is a sequence-specific DNA-binding protein that serves to regulate both transcription and DNA replication. E1, a DNA helicase, is the only PV protein that possesses enzymatic activity (5) and is also the most highly conserved of the PV proteins. For these reasons, E1 has been considered as the most attractive molecular target for the development of antiviral agents (6).During the initiation of PV DNA replication, E2 serves as a specificity factor to enhance binding of E1 monomers to the origin, which by themselves bind to double-stranded DNA with little sequence specificity (4, 7-11). E2 dimers bind with high specificity to DNA sequences within the origin and recruit to it E1 monomers, through direct protein-protein interactions (12, 13). Upon binding to DNA, E1 monomers assemble into hexamers (14, 15), but the E1-E2 interaction is not maintained (16,17). Thus, the role of E2 is to catalyze the assembly of hexameric E1 complexes specifically at the origin. ATP hydrolysis is also required for E1 hexamers to distort the orig...

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“…(RepA) 6 is one of the smallest known DNA-helicases with a molecular mass of 6U29 896 kDa [4,5]. In contrast to other multimeric helicases which require Mg 2 , ATP or ssDNA to assemble into multimeric forms, the hexameric structure of (RepA) 6 is stable even in the absence of any cofactor.…”

Section: Introductionmentioning

confidence: 99%

“…Depending on the pH and cofactors present, DnaB-helicase can form a triangle-shaped oligomer built by a trimer of dimers [2]. Bacteriophage T7 gene 4 helicase and plasmid RSF1010 encoded hexameric RepA helicase ((RepA) 6 ) are also ring-shaped hexamers [3,4]. These helicases have in common a central channel through which single-stranded DNA (ssDNA) is assumed to pass.…”

Section: Introductionmentioning

confidence: 99%

“…Since one ssDNA is assumed to pass through the central channel of ring-shaped helicases during the unwinding reaction while the second ssDNA strand moves along the outsite of the helicase mole-cule, the question arises how many binding sites for ssDNA molecules are present on hexameric helicases. To answer this question, we measured the dissociation constants, K d , and the stoichiometries, n, for the binding of (dA) 30 to puri¢ed (RepA) 6 at optimum and minimum helicase activity at pH 5.8 and 7.6 [4,5], respectively, by using analytical ultracentrifugation.…”

Section: Introductionmentioning

confidence: 99%

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Interaction of different oligomeric states of hexameric DNA‐helicase RepA with single‐stranded DNA studied by analytical ultracentrifugation

Frank

Holzwarth

et al. 2000

FEBS Letters

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Analytical ultracentrifugation was used to determine the molecular mass, M, of hexameric DNA-helicase RepA at pH 5.8 and 7.6. At pH 7.6, a molecular mass of 179.5 þ 2.6 kDa was found, consistent with the known hexameric state of RepA, (RepA) 6 . At pH 5.8, (RepA) 6 associates to form a dimer with a molecular mass of 366.2 þ 4.1 kDa. Analytical ultracentrifugation was also applied to characterize the interaction of singlestranded DNA (ssDNA) with the two different oligomeric states of (RepA) 6 at pH 5.8 and 7.6. The dissociation constants, K d , for the equilibrium binding of (dA) 30 to the (RepA) 6 dimer at pH 5.8 and to (RepA) 6 at pH 7.6 were determined at 10³C in the presence of 0.5 mM ATPQ QS, 10 mM MgCl 2 and 60 mM NaCl as K d5X8 = 0.94 þ 0.13 W WM at pH 5.8 and K d7X6 = 25.4 þ 6.4 W WM at pH 7.6. The stoichiometries, n, for the two complexes (dA) 30 / (RepA) 6 dimer and (dA) 30 /(RepA) 6 at pH 5.8 and 7.6 were calculated from the corresponding binding curves. At pH 5.8 one (dA) 30 molecule was bound per (RepA) 6 dimer, while at pH 7.6 one (dA) 30 molecule was bound to one (RepA) 6 . Binding curves were compatible with a single ssDNA binding site present on the (RepA) 6 dimer and on (RepA) 6 , respectively, with no indication of cooperativity. (RepA) 6 tends to form larger aggregates under acidic conditions (pH 6 6.0) which are optimal for ssDNA binding. In contrast, at pH 5.8 in the presence of 60 mM NaCl, only the (RepA) 6 dimer was observed both in the absence and presence of (dA) 30 . ß

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The RepA Protein of Plasmid RSF1010 Is a Replicative DNA Helicase

Cited by 56 publications

References 61 publications

Sequence-related protein export NTPases encoded by the conjugative transfer region of RP4 and by the cag pathogenicity island of Helicobacter pylori share similar hexameric ring structures

Sequence-related protein export NTPases encoded by the conjugative transfer region of RP4 and by the cag pathogenicity island of Helicobacter pylori share similar hexameric ring structures

Characterization of Recombinant HPV6 and 11 E1 Helicases

Interaction of different oligomeric states of hexameric DNA‐helicase RepA with single‐stranded DNA studied by analytical ultracentrifugation

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