DNA Helicase RepA:  Cooperative ATPase Activity and Binding of Nucleotides

Xu, Hai; Frank, Joachim; Niedenzu, Timo

doi:10.1021/bi0008938

Cited by 15 publications

(19 citation statements)

References 46 publications

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“…An open and a more closed conformation are arranged in alternating positions in the cubic structure of RepA, demonstrating the existence of three equivalent dimers in the hexameric ring structure associated with two types of ATP binding sites. This ®nding is also in agreement with biochemical studies which showed that the ATP binding sites in RepA are not identical but three are of low and three of high af®nity for ATP (34). Mechanistically this is probably important because the conversion of the open into the closed type adenine binding site may lead to some kind of a pulsation of the molecule that could periodically change the diameter or the shape of the central hole.…”

Section: Mechanistic Implications Of the Two Conformations Observed At The Subunit Interfacessupporting

confidence: 88%

Hexameric RSF1010 helicase RepA: the structural and functional importance of single amino acid residues

Ziegelin

Niedenzu²,

Lanka

2003

Nucleic Acids Research

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In the known monoclinic crystals the 3-dimensional structure of the hexameric, replicative helicase RepA encoded by plasmid RSF1010 shows 6-fold rotational symmetry. In contrast, in the cubic crystal form at 2.55 A resolution described here RepA has 3-fold symmetry and consists of a trimer of dimers. To study structure-function relationships, a series of repA deletion mutants and mutations yielding single amino acid exchanges were constructed and the respective gene products were analyzed in vivo and in vitro. Hexamerization of RepA occurs via the N-terminus and is required for NTP hydrolysis. The C-terminus is essential both for the interaction with the replication machinery and for the helicase activity. Functional analyses of RepA variants with single amino acid exchanges confirmed most of the predictions that were based on the published 3-dimensional structure. Of the five motifs conserved in family 4 helicases, all residues conserved in RepA and T7 gp4 helicases participate in DNA unwinding. Residues K42, E76, D77, D139 and H178, proposed to play key roles in catalyzing the hydrolysis of NTPs, are essential for RepA activity. Residue H178 of motif H3 couples nucleotide consumption to DNA strand separation.

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Section: Mechanistic Implications Of the Two Conformations Observed At The Subunit Interfacessupporting

confidence: 88%

Hexameric RSF1010 helicase RepA: the structural and functional importance of single amino acid residues

Ziegelin

Niedenzu²,

Lanka

2003

Nucleic Acids Research

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“…The RepA protein is a hexameric DNA replicative helicase that is essential for replication of the RFS1010 plasmid, a broad host nonconjugative plasmid that can replicate in most Gram-negative bacteria and confers bacterial resistance to sulfonamides and streptomycin (accompanying paper 1,[1][2][3][4][5][6][7][8]. The protein forms a very stable, ring-like hexameric structure in the absence of any cofactors or salt in solution (3,5,6).…”

mentioning

confidence: 99%

Binding of Six Nucleotide Cofactors to the Hexameric Helicase RepA Protein of Plasmid RSF1010. 2. Base Specificity, Nucleotide Structure, Magnesium, and Salt Effect on the Cooperative Binding of the Cofactors

2005

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Interactions of the RepA hexameric helicase with nucleotide cofactors have been examined using nucleotide analogues, TNP-ADP and TNP-ATP, and unmodified nucleotides. Thermodynamic parameters for the interactions of modified and unmodified nucleotides have been obtained using quantitative fluorescence titration and competition titration methods. The intrinsic binding constant of ATP is by a factor of approximately 10 and approximately 1000 higher than the value observed for ADP and PO(4)(-). The data suggest that helicase acquires free-energy transducing capabilities when associated with the ssDNA, thus, forming a "holoenzyme". ATP binding is characterized by significantly stronger negative cooperativity than ADP. The cooperative interactions are predominantly induced through the specific interactions of the gamma phosphate and the ribose with the protein. The salt effect on cofactor binding indicates a very different nature of the intrinsic and cooperative interactions. Surprisingly, binding of Mg(2+), to both the cofactor and helicase, predominantly controls the ADP-RepA interactions. Mg(2+) cations seem to play a role in affecting the distribution of high and low ssDNA-affinity states, through the strong effect on the diphosphate versus triphosphate binding. The data indicate that Mg(2+) has a dual function in nucleotide-helicase interactions. At low [Mg(2+)], NTP binds stronger than NDP and the enzyme is predominantly in the high ssDNA-affinity state. At higher [Mg(2+)], NTP binds weaker than NDP and the helicase subunits can exist in alternating low- and high-affinity states that facilitate the efficient dsDNA unwinding. The RepA helicase shows a preference toward purine nucleotides. The cooperative interactions are independent of the type of the base.

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“…For rotating motors, their channel diameter should be no larger than 2 nm (the diameter of a dsDNA) to allow for close contact between a DNA and the channel wall for threading, since a ssDNA within the channel displays an A form helical structure and is smaller than 2 nm in diameter . Examples include rotating motors of DnaB, Rho factor, − TrwB, − MCM, and RepA or RuvB, − all of which have a channel diameter of 1–2 nm …”

Section: Revolving and Rotating Motors Can Be Distinguished By Their ...mentioning

confidence: 99%

“…Revolving rather than rotating avoids the coiling and tangling of long polymer chains, such as genomic dsDNAs during translocation. The well-studied rotating motors include F1/F0 ATPase, − DNA helicase, , Rho transcription termination factor, − TrwB, − MCM, , and RepA or RuvB, − all of which have a channel diameter of 1–2 nm . Revolving motors include the DNA translocases Ftsk in Gram-negative bacteria, SpoIIIE or SftA (YtpS) in Gram-positive bacteria, A32 ATPase of poxvirus, − DNA packaging enzyme of adenovirus, − the genome segregation enzymes of mimivirus, ,− as well as the DNA packaging motors of herpesvirus, − SPP1, T7, HK97, P22, and Phi29 .…”

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confidence: 99%

Controlling the Revolving and Rotating Motion Direction of Asymmetric Hexameric Nanomotor by Arginine Finger and Channel Chirality

et al. 2019

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Nanomotors in nanotechnology are as important as engines in daily life. Many ATPases are nanoscale biomotors classified into three categories based on the motion mechanisms in transporting substrates: linear, rotating, and the recently discovered revolving motion. Most biomotors adopt a multisubunit ring-shaped structure that hydrolyzes ATP to generate force. How these biomotors control the motion direction and regulate the sequential action of their multiple subunits is intriguing. Many ATPases are hexameric with each monomer containing a conserved arginine finger. This review focuses on recent findings on how the arginine finger controls motion direction and coordinates adjacent subunit interactions in both revolving and rotating biomotors. Mechanisms of intersubunit interactions and sequential movements of individual subunits are evidenced by the asymmetrical appearance of one dimer and four monomers in high-resolution structural complexes. The arginine finger is situated at the interface of two subunits and extends into the ATP binding pocket of the downstream subunit. An arginine finger mutation results in deficiency in ATP binding/hydrolysis, substrate binding, and transport, highlighting the importance of the arginine finger in regulating energy transduction and motor function. Additionally, the roles of channel chirality and channel size are discussed as related to controlling one-way trafficking and differentiating the revolving and rotating mechanisms. Finally, the review concludes by discussing the conformational changes and entropy conversion triggered by ATP binding/hydrolysis, offering a view different from the traditional concept of ATP-mediated mechanochemical energy coupling. The elucidation of the motion mechanism and direction control in ATPases could facilitate nanomotor fabrication in nanotechnology.

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DNA Helicase RepA: Cooperative ATPase Activity and Binding of Nucleotides

Cited by 15 publications

References 46 publications

Hexameric RSF1010 helicase RepA: the structural and functional importance of single amino acid residues

Hexameric RSF1010 helicase RepA: the structural and functional importance of single amino acid residues

Binding of Six Nucleotide Cofactors to the Hexameric Helicase RepA Protein of Plasmid RSF1010. 2. Base Specificity, Nucleotide Structure, Magnesium, and Salt Effect on the Cooperative Binding of the Cofactors

Controlling the Revolving and Rotating Motion Direction of Asymmetric Hexameric Nanomotor by Arginine Finger and Channel Chirality

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