Protein surface salt bridges and paths for DNA wrapping

Saecker, Ruth M.; Record, M. Thomas

doi:10.1016/s0959-440x(02)00326-3

Cited by 67 publications

(87 citation statements)

References 62 publications

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“…At low KCl concentration both ΔH obs and ΔC p,obs are large and negative, but their magnitudes decrease non-linearly as the salt concentration increases. These results, which are similar to those reported here and previously (15), were interpreted as reflecting the DNA binding dependent disruption of salt bridges on the protein surface formed between cationic (Lys, Arg and His) and anionic (Asp and Glu) residues (25,80). According to this model, at low salt concentrations the salt bridges on the protein are mainly intact in the free protein, but are disrupted by DNA binding, resulting in hydration of both cationic and anionic side chains prior to DNA binding.…”

Section: Nature Of the Weak Interactions Of Anions With Ssb Manifestesupporting

confidence: 92%

Effects of Monovalent Anions on a Temperature-Dependent Heat Capacity Change for Escherichia coli SSB Tetramer Binding to Single-Stranded DNA

Козлов

Lohman

2006

Biochemistry

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We have previously shown that the linkage of temperature-dependent protonation and DNA base unstacking equilibria contribute significantly to both the negative enthalpy change (ΔH obs ) and the negative heat capacity change (ΔC p,obs ) for E. coli SSB homotetramer binding to single stranded (ss) DNA. Using isothermal titration calorimetry we have now examined ΔH obs over a much wider temperature range (5°C to 60°C) and as a function of monovalent salt concentration and type for SSB binding to (dT) 70 under solution conditions that favor the fully wrapped (SSB) 65 complex (monovalent salt concentration ≥ 0.20 M). Over this wider temperature range we observe a strongly temperature dependent ΔC p,obs . The ΔH obs decreases as temperature increases from 5°C to 35°C (ΔC p,obs <0), but then increase at higher temperatures up to 60°C (ΔC p,obs >0). Both salt concentration and anion type have large effects on ΔH obs and ΔC p,obs . These observations can be explained by a model in which SSB protein can undergo a temperature and salt dependent conformational transition (below 35°C), the midpoint of which shifts to higher temperature (above 35°C) for SSB bound to ssDNA. Anions bind weakly to free SSB, with the preference, Br -> Cl -> F -, and these anions are then released upon binding ssDNA, affecting both ΔH obs and ΔC p,obs . We conclude that the experimentally measured values of ΔC p,obs for SSB binding to ssDNA cannot be explained solely on the basis of changes in accessible surface area (ASA) upon complex formation, but rather result from a series of temperature dependent equilibria (ion binding, protonation and protein conformational changes) that are coupled to the SSB-ssDNA binding equilibrium. This is also likely true for many other protein-nucleic acid interactions.Obtaining an understanding of the molecular origins for stability and specificity of proteinnucleic acid interactions is a complex problem. Such interactions not only involve multiple networks of hydrogen bonds, salt bridges and non-polar interactions, but are also accompanied by the coupled binding and/or release of small molecules, such as protons, salt ions and water. These coupled binding equilibria will generally contribute significantly to the thermodynamics of protein-nucleic acid binding (ΔG°, ΔH° and ΔS°). For this reason, thermodynamic information obtained under only one set of solution conditions is generally of limited utility for understanding the origins of stability and specificity of any macromolecular interaction, including protein-nucleic acid binding. † This research was supported in part by the NIH (R01 GM30498) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptWe have been studying the thermodynamics of E. coli SSB protein binding to single stranded (ss) nucleic acids (for reviews see (1-3)), a protein that is involved in DNA replication, recombination and repair (4). E.coli SSB protein is a stable homotetramer (4×18,843 Da) (5), that can interact with ssDNA in multiple binding modes, depending on...

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Section: Nature Of the Weak Interactions Of Anions With Ssb Manifestesupporting

confidence: 92%

Effects of Monovalent Anions on a Temperature-Dependent Heat Capacity Change for Escherichia coli SSB Tetramer Binding to Single-Stranded DNA

Козлов

Lohman

2006

Biochemistry

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“…This surface groove contains pairs of conserved cationic (␤Ј R576, R610, K615; ␣ H128) and anionic (␤Ј D571 and E616) residues (see SI Methods) positioned to be able to form intramolecular salt bridges in the absence of DNA, and hence modulate the affinity of RNAP for the upstream region of promoter DNA (27). Although the ⅐OH backbone protection data do not precisely define the phasing of the far-upstream DNA with respect to RNAP (see SI Methods), they unambiguously demonstrate that the ''backside'' of RNAP interacts extensively with this DNA in I 1 , but not in RP o .…”

Section: Comparison Of the Extent Of Dna Opening In Rpo With I1 Usingmentioning

confidence: 99%

Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase

Davis¹,

Bingman

Landick³

et al. 2007

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

197

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initiation ͉ transcription ͉ wrapping ͉ kinetics S pecific transcription initiation by Escherichia coli RNA polymerase (RNAP: core subunit composition ␣ 2 ␤␤Ј ϩ 70 ϭ holoenzyme) at promoter sequences is determined by recognition of DNA (Ϫ10 and Ϫ35 hexamers) upstream of the start site (ϩ1) by the specificity subunit 70 . Subsequent to binding, a series of large-scale conformational changes in both RNAP and promoter DNA create the initiation-competent open complex (RP o ) (1). During these steps, the multisubunit bacterial RNAP acts as an intricate molecular machine and opens Ϸ14 bp of the DNA double helix. Defining the cascade of conformational changes that occur during initiation is essential to understand sequence-and factordependent regulation of the rate of transcription initiation and has important applications in chemical biology and in antibiotic design. However, the intermediates on this pathway are relatively unstable and short-lived and hence are difficult to trap unambiguously. To date, all structural information about complexes known to be on-pathway intermediates in RP o formation has come from chemical and enzymatic DNA footprinting methods.Quantitative kinetic-mechanistic studies find that at least two kinetically significant intermediates, generically designated I 1 and I 2 , precede formation of RP o by E. coli RNAP:where the relatively slow interconversions between I 1 and I 2 are rate-limiting in both the forward and back directions (2, 3). In the mechanism shown in Eq. 1, I 2 and RP o are characterized by their resistance to a short challenge with a polyanionic competitor such as heparin, which acts to sequester any free RNAP present during the challenge. (In contrast, after a 10 to 20 sec challenge with heparin, I 1 complexes, which are in rapid equilibrium with free RNAP and promoter sequences, are eliminated from the population.) Given the high degree of conservation of bacterial RNAP and promoter DNA sequences, this mechanism is likely to describe the key steps in initiation in most prokaryotes. Moreover, conservation of many elements of sequence, structure, and/or function between bacterial and eukaryotic polymerase (pol II) subunits and transcription factors supports the inference that the bacterial intermediates may be homologs of initiation intermediates formed by pol II (4, 5).Recently we (6) and Ross and Gourse (7) found that the presence of DNA upstream of the Ϫ35 promoter recognition hexamer greatly accelerates (up to Ϸ60-fold) the rate-determining isomerization step (conversion of I 1 to I 2 ). Strikingly, DNase I footprinting of I 1 at the strong bacteriophage promoter P R reveals that when nonspecific DNA upstream of base pair Ϫ47 is present, downstream DNA is protected to around ϩ20, and thus bound in the active-site channel of RNAP. However, when DNA upstream of Ϫ47 is deleted,

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“…In this way, the C-terminal tail of the RecA protein could form a flap that regulates accessibility to the nucleoprotein filament. The coupling of protein surface salt bridge disruption to DNA binding is a common mechanism used by DNA-binding proteins and has been reviewed recently (54).…”

Section: Mgmentioning

confidence: 99%

Magnesium Ion-dependent Activation of the RecA Protein Involves the C Terminus

Lusetti

Shaw

Cox

2003

Journal of Biological Chemistry

122

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Optimal conditions for RecA protein-mediated DNA strand exchange include 6 -8 mM Mg 2؉ in excess of that required to form complexes with ATP. We provide evidence that the free magnesium ion is required to mediate a conformational change in the RecA protein C terminus that activates RecA-mediated DNA strand exchange. In particular, a "closed" (low Mg 2؉ ) conformation of a RecA nucleoprotein filament restricts DNA pairing by incoming duplex DNA, although singlestranded overhangs at the ends of a duplex allow limited DNA pairing to occur. The addition of excess Mg 2؉ results in an "open" conformation, which can promote efficient DNA pairing and strand exchange regardless of DNA end structure. The removal of 17 amino acid residues at the Escherichia coli RecA C terminus eliminates a measurable requirement for excess Mg 2؉ and permits efficient DNA pairing and exchange similar to that seen with the wild-type protein at high Mg 2؉ levels. Thus, the RecA C terminus imposes the need for the high magnesium ion concentrations requisite in RecA reactions in vitro. We propose that the C terminus acts as a regulatory switch, modulating the access of double-stranded DNA to the presynaptic filament and thereby inhibiting homologous DNA pairing and strand exchange at low magnesium ion concentrations.The RecA protein of Escherichia coli plays a central role in the processes of homologous DNA recombination and DNA repair. RecA is a DNA-dependent ATPase that catalyzes an in vitro DNA strand exchange reaction between single-stranded (ssDNA) 1 and homologous double-stranded DNA (dsDNA) molecules. The DNA strand exchange reaction takes place in several stages (Fig. 1). The RecA protein forms a nucleoprotein filament that completely encompasses the circular ssDNA. This filament then aligns the bound single strand with a homologous duplex DNA to form a DNA pairing intermediate often referred to as a joint molecule. 1000 base pairs of DNA can be aligned and exchanged in a joint molecule under the empirically defined optimal reaction conditions, which typically include 1-3 mM ATP and about 10 mM magnesium ion. All steps to this point, including the formation of joint molecules, require ATP but not ATP hydrolysis. ATP hydrolysis is needed only to complete the late stages of strand exchange of long DNA substrates, often derived from bacteriophage DNAs. Whereas DNA pairing, leading to joint molecule formation, can occur at either end of a linear duplex, the subsequent and ATP hydrolysisdependent extension of the nascently paired regions is unidirectional, proceeding 5Ј to 3Ј relative to ssDNA initially bound in the filament. Thus, exchange proceeds in one direction along the linear duplex, and joints formed at the "wrong" end in the pairing phase are eliminated. In a DNA strand exchange involving quite long DNAs that leads to nicked circular product formation, the ends of the duplex where the exchange begins and ends are referred to as proximal and distal, respectively ( Fig. 1) (1, 2).Examination of the conditions for an optimal RecA prot...

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Protein surface salt bridges and paths for DNA wrapping

Cited by 67 publications

References 62 publications

Effects of Monovalent Anions on a Temperature-Dependent Heat Capacity Change for Escherichia coli SSB Tetramer Binding to Single-Stranded DNA

Effects of Monovalent Anions on a Temperature-Dependent Heat Capacity Change for Escherichia coli SSB Tetramer Binding to Single-Stranded DNA

Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase

Magnesium Ion-dependent Activation of the RecA Protein Involves the C Terminus

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