The Escherichia coli PriA Helicase Specifically Recognizes Gapped DNA Substrates

Szymanski, Michal R.; Jezewska, Maria J.; Bujalowski, Wlodzimierz

doi:10.1074/jbc.m109.094789

Cited by 12 publications

(39 citation statements)

References 59 publications

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“…Adding a synthetic nascent leading strand increases PriA affinity for the DNA fork and orients PriA to preferentially unwind the nascent lagging strand (39,40). The nascent lagging strand is 5 nt shorter than the template non-complementary region, leaving a 5 nt gap on the lagging strand adjacent to the fork junction (29) that enhances PriA helicase activity ((39,41) and data not shown). PriA preferential unwinding of the nascent lagging strand in this substrate can be stimulated by the addition of either SSB or PriB (29,33,38,39).…”

Section: Resultsmentioning

confidence: 99%

An aromatic-rich loop couples DNA binding and ATP hydrolysis in the PriA DNA helicase

Windgassen¹,

Keck²

2016

Nucleic Acids Res

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Helicases couple ATP hydrolysis to nucleic acid binding and unwinding via molecular mechanisms that remain poorly defined for most enzyme subfamilies within the superfamily 2 (SF2) helicase group. A crystal structure of the PriA SF2 DNA helicase, which governs restart of prematurely terminated replication processes in bacteria, revealed the presence of an aromatic-rich loop (ARL) on the presumptive DNA-binding surface of the enzyme. The position and sequence of the ARL was similar to loops known to couple ATP hydrolysis with DNA binding in a subset of other SF2 enzymes, however, the roles of the ARL in PriA had not been investigated. Here, we show that changes within the ARL sequence uncouple PriA ATPase activity from DNA binding. In vitro protein-DNA crosslinking experiments define a residue- and nucleotide-specific interaction map for PriA, showing that the ARL binds replication fork junctions whereas other sites bind the leading or lagging strands. We propose that DNA binding to the ARL allosterically triggers ATP hydrolysis in PriA. Additional SF2 helicases with similarly positioned loops may also couple DNA binding to ATP hydrolysis using related mechanisms.

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Section: Resultsmentioning

confidence: 99%

An aromatic-rich loop couples DNA binding and ATP hydrolysis in the PriA DNA helicase

Windgassen¹,

Keck²

2016

Nucleic Acids Res

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“…To overcome the problem of resorting to complex numerical calculations, one can apply a combined application of the generalized McGheevon Hippel model, as defined by Equations 8 and 9, and the combinatorial theory for large ligand binding to a linear, homogeneous lattice (31,32,36,38). In this approach, the ligand binding to the reference fluorescent nucleic acid is described by the generalized McGhee-von Hippel equation.…”

Section: Base Specificity Of Denv Polymerase-ssdna Interactions; Lattmentioning

confidence: 99%

“…In studies described in this work, we use, as a reference lattice, poly(⑀A), and the base specificity of the DENV polymerase has been examined using different ssRNA homopolymers, poly(A), poly(U), and poly(C). At a given titration point, i, the total concentration of the bound protein, P b , is defined as follows (31,32,36,38),…”

Section: Base Specificity Of Denv Polymerase-ssdna Interactions; Lattmentioning

confidence: 99%

“…Thus, both 30-mers should bind only a single molecule of the polymerase, which greatly simplifies the binding analysis. The reference fluorescent nucleic acid is poly(⑀A) (31,32,36,38). In the presence of the unmodified 30-mer, at a given titration point, i, the total concentration of the bound protein, P b , is defined by an expression analogous to Equation 10, as follows, where (⌺v i ) R and (⌺⍜ i ) S are the total average binding density of the DENV polymerase on the reference poly(⑀A) and the total average degree of binding on the examined unmodified 30-mer, respectively.…”

Section: Base Specificity Of Denv Polymerase-ssdna Interactions; Lattmentioning

confidence: 99%

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Full-length Dengue Virus RNA-dependent RNA Polymerase-RNA/DNA Complexes

Szymanski

Jezewska

Bujalowski

et al. 2011

Journal of Biological Chemistry

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Fundamental aspects of interactions of the Dengue virus type 3 full-length polymerase with the single-stranded and doublestranded RNA and DNA have been quantitatively addressed. The polymerase exists as a monomer with an elongated shape in solution. In the absence of magnesium, the total site size of the polymerase-ssRNA complex is 26 ؎ 2 nucleotides. In the presence of Mg 2؉ , the site size increases to 29 ؎ 2 nucleotides, indicating that magnesium affects the enzyme global conformation. The enzyme shows a preference for the homopyrimidine ssRNAs. Positive cooperativity in the binding to homopurine ssRNAs indicates that the type of nucleic acid base dramatically affects the enzyme orientation in the complex. Both the intrinsic affinity and the cooperative interactions are accompanied by a net ion release. The polymerase binds the dsDNA with an affinity comparable with the ssRNAs affinity, indicating that the binding site has an open conformation in solution. The lack of detectable dsRNA or dsRNA-DNA hybrid affinities indicates that the entry to the binding site is specific for the sugar-phosphate backbone and/or conformation of the duplex. The dengue virus (DENV)2 is a member of the Flaviviridae family and the etiological agent of dengue fever, a disease affecting worldwide ϳ100 million people each year (1-5). The Flaviviridae family includes such human pathogens as tick-borne encephalitis virus, hepatitis C virus, West Nile virus, yellow fever virus, and Japanese encephalitis virus (1-5). The dengue virus exists in four distinct types, DENV1, -2, -3, and -4, which show ϳ60% genomic sequence identity (1). The dengue fever often develops into the dengue hemorrhagic fever, an acute form of the disease, possibly as a result of sequential infection with different types of the virus. It is characterized by a significant mortality, particularly for individuals with a weakened or undeveloped immune system. The small, 10.7-kbp positive strand RNA genome of the dengue virus encodes only 10 proteins: three structural proteins (capsid, premembrane, and envelope proteins) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 proteins) (3, 6 -8). The entire viral genome is translated as a single polyprotein, subsequently cleaved by viral and cellular proteases into functional entities.The NS5 protein is the viral replicative RNA-dependent RNA polymerase (1, 3, 6 -8). The primary structure of the fulllength polymerase encompasses 900 amino acids (i.e. it is the largest of the nonstructural proteins of the dengue virus) (6 -10). The enzyme is built of two functional domains (6 -10). The N-terminal domain includes the first 263 amino acids. It functions as a methyltransferase (MTase), whose activity is necessary for viral RNA recognition by the host cell translational apparatus. Correspondingly, amino acids ϳ273-906 form the polymerase domain, containing the active site of the RNA synthesis (Fig. 1a) (6 -8). The crystal structures of the isolated N-terminal and polymerase domains have been determined up t...

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“…Activities of the PriA protein in vivo are related to the ability of the enzyme to interact with both the ss and the dsDNA (1–12,15–17,19–22). Quantitative thermodynamic analyses showed that in the complex with the ssDNA, the total DNA-binding site of the PriA helicase occludes ~20 nucleotides of the nucleic acid (15–17,19–22).…”

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confidence: 99%

The N-Terminal Domain of the Escherichia coli PriA Helicase Contains Both the DNA- and Nucleotide-Binding Sites. Energetics of Domain–DNA Interactions and Allosteric Effect of the Nucleotide Cofactors

et al. 2011

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Functional interactions of the E. coli PriA helicase 181N-terminal domain with the DNA and nucleotide cofactors have been quantitatively examined. The isolated 181N-terminal domain forms a stable dimer in solution, most probably reflecting the involvement of the domain in specific cooperative interactions of the intact PriA protein - dsDNA complex. Only one monomer of the domain dimer binds the DNA, i.e., the dimer has one effective DNA-binding site. Although the total site-size of the dimer - ssDNA complex is ~13 nucleotides, the DNA-binding subsite engages in direct interactions ~5 nucleotides. A small number of interacting nucleotides indicates that the DNA-binding subsites of the PriA helicase, i.e., the strong subsite on the helicase domain and the weak subsite on the N-terminal domain, are spatially separated in the intact enzyme. Contrary to current views, the subsite has only a slight preference for the 3′-end OH group of the ssDNA and lacks any significant base specificity, although it has a significant dsDNA affinity. Unlike the intact helicase, the DNA-binding subsite of the isolated domain is in an open conformation, indicating the presence of the direct helicase domain - N-terminal domain interactions. The discovery that the 181N-terminal domain possesses a nucleotide-binding site places the allosteric, weak nucleotide-binding site of the intact PriA on the N-terminal domain. The specific ADP effect on the domain DNA-binding subsite indicates that in the intact helicase, the bound ADP not only opens the DNA-binding subsite but also increases its intrinsic DNA affinity.

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The Escherichia coli PriA Helicase Specifically Recognizes Gapped DNA Substrates

Cited by 12 publications

References 59 publications

An aromatic-rich loop couples DNA binding and ATP hydrolysis in the PriA DNA helicase

An aromatic-rich loop couples DNA binding and ATP hydrolysis in the PriA DNA helicase

Full-length Dengue Virus RNA-dependent RNA Polymerase-RNA/DNA Complexes

The N-Terminal Domain of the Escherichia coli PriA Helicase Contains Both the DNA- and Nucleotide-Binding Sites. Energetics of Domain–DNA Interactions and Allosteric Effect of the Nucleotide Cofactors

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