The Escherichia coli PriA Helicase-Double-Stranded DNA Complex: Location of the Strong DNA-Binding Subsite on the Helicase Domain of the Protein and the Affinity Control by the Two Nucleotide-Binding Sites of the Enzyme

The Escherichia coli PriA helicase complex with the double-stranded DNA (dsDNA), the location of the strong DNA-binding subsite, and the effect of the nucleotide cofactors, bound to the strong and weak nucleotide-binding site of the enzyme on the dsDNA affinity, have been analyzed using the fluorescence titration, analytical ultracentrifugation, and photo-cross-linking techniques. The total site size of the PriA-dsDNA complex is only 5 ± 1 bp, that is, dramatically lower than 20 ± 3 nucleotides occluded in the enzyme-single-stranded DNA (ssDNA) complex. The helicase associates with the dsDNA using its strong ssDNA-binding subsite in an orientation very different from the complex with the ssDNA. The strong DNA-binding subsite of the enzyme is located on the helicase domain of the PriA protein. The dsDNA intrinsic affinity is considerably higher than the ssDNA affinity and the binding process is accompanied by a significant positive cooperativity. Association of cofactors with strong and weak nucleotide-binding sites of the protein profoundly affects the intrinsic affinity and the cooperativity, without affecting the stoichiometry. ATP analog binding to either site diminishes the intrinsic affinity but preserves the cooperativity. ADP binding to the strong site leads to a dramatic increase of the cooperativity and only slightly affects the affinity, while saturation of both sites with ADP strongly increases the affinity and eliminates the cooperativity. Thus, the coordinated action of both nucleotide-binding sites on the PriA-dsDNA interactions depends on the structure of the phosphate group. The significance of these results for the enzyme activities in recognizing primosome assembly sites or the ssDNA gaps is discussed.     

Electrochemical detection of mismatched DNA using a MutS probe

A direct and label-free electrochemical biosensor for the detection of the protein–mismatched DNA interaction was designed using immobilized N-terminal histidine tagged Escherichia coli. MutS on a Ni-NTA coated Au electrode. General electrochemical methods, cyclic voltammetry (CV), electrochemical quartz crystal microbalance (EQCM) and impedance spectroscopy, were used to ascertain the binding affinity of mismatched DNAs to the MutS probe. The direct results of CV and impedance clearly reveal that the interaction of MutS with the CC heteroduplex was much stronger than that with AT homoduplex, which was not differentiated in previous results (GT > CT > CC ≈ AT) of a gel mobility shift assay. The EQCM technique was also able to quantitatively analyze MutS affinity to heteroduplexes.     

Steady-state ATPase activity of E. coli MutS modulated by its dissociation from heteroduplex DNA

The ability of MutS to recognize mismatched DNA is required to initiate a mismatch repair (MMR) system. ATP binding and hydrolysis are essential in this process, but their role in MMR is still not fully understood. In this study, steady-state ATPase activities of MutS from Escherichia coli were investigated using the spectrophotometric method with a double end-blocked heteroduplex containing gapped bases. The ATPase activities of MutS increased as the number of gapped bases increased in a double end-blocked heteroduplex with 2-8 gapped bases in the chain, indicating that MutS dissociates from DNA when it reaches a scission during movement along the DNA. Since movement of MutS along the chain does not require extensive ATP hydrolysis and the ATPase activity is only enhanced when MutS dissociates from a heteroduplex, these results support the sliding clamp model in which ATP binding by MutS induces the formation of a hydrolysis-independent sliding clamp.     

Structural and functional analysis of the MutS C-terminal tetramerization domain

The Escherichia coli DNA mismatch repair (MMR) protein MutS is essential for the correction of DNA replication errors. In vitro, MutS exists in a dimer/tetramer equilibrium that is converted into a monomer/dimer equilibrium upon deletion of the C-terminal 53 amino acids. In vivo and in vitro data have shown that this C-terminal domain (CTD, residues 801–853) is critical for tetramerization and the function of MutS in MMR and anti-recombination. We report the expression, purification and analysis of the E.coli MutS-CTD. Secondary structure prediction and circular dichroism suggest that the CTD is folded, with an α-helical content of 30%. Based on sedimentation equilibrium and velocity analyses, MutS-CTD forms a tetramer of asymmetric shape. A single point mutation (D835R) abolishes tetramerization but not dimerization of both MutS-CTD and full-length MutS. Interestingly, the in vivo and in vitro MMR activity of MutS^CF/D835R is diminished to a similar extent as a truncated MutS variant (MutS800, residues 1–800), which lacks the CTD. Moreover, the dimer-forming MutS^CF/D835R has comparable DNA binding affinity with the tetramer-forming MutS, but is impaired in mismatch-dependent activation of MutH. Our data support the hypothesis that tetramerization of MutS is important but not essential for MutS function in MMR.     

Structural insight into the mechanism of substrate specificity and catalytic activity of an HD-domain phosphohydrolase: the 5'-deoxyribonucleotidase YfbR from Escherichia coli

HD-domain phosphohydrolases have nucleotidase and phosphodiesterase activities and play important roles in the metabolism of nucleotides and in signaling. We present three 2.1-Å-resolution crystal structures (one in the free state and two complexed with natural substrates) of an HD-domain phosphohydrolase, the Escherichia coli 5′-nucleotidase YfbR. The free-state structure of YfbR contains a large cavity accommodating the metal-coordinating HD motif (H33, H68, D69, and D137) and other conserved residues (R18, E72, and D77). Alanine scanning mutagenesis confirms that these residues are important for activity. Two structures of the catalytically inactive mutant E72A complexed with Co²⁺ and either thymidine-5′-monophosphate or 2′-deoxyriboadenosine-5′-monophosphate disclose the novel binding mode of deoxyribonucleotides in the active site. Residue R18 stabilizes the phosphate on the Co²⁺, and residue D77 forms a strong hydrogen bond critical for binding the ribose. The indole side chain of W19 is located close to the 2′-carbon atom of the deoxyribose moiety and is proposed to act as the selectivity switch for deoxyribonucleotide, which is supported by comparison to YfdR, another 5′-nucleotidase in E. coli. The nucleotide bases of both deoxyriboadenosine-5′-monophosphate and thymidine-5′-monophosphate make no specific hydrogen bonds with the protein, explaining the lack of nucleotide base selectivity. The YfbR E72A substrate complex structures also suggest a plausible single-step nucleophilic substitution mechanism. This is the first proposed molecular mechanism for an HD-domain phosphohydrolase based directly on substrate-bound crystal structures.     

ATP-hydrolysis-dependent conformational switch modulates the stability of MutS-mismatch complexes

The mismatch repair pathway in Escherichia coli has been extensively studied in vitro as well as in vivo. The molecular mechanisms by which nucleotide cofactors regulate the whole process constitute an area of active debate. Here we demonstrate that nucleotide (ADP or ATP) binding to MutS mediates a switch in protein conformation. However, in MutS that is DNA bound, this switch ensues only with ATP and not with ADP and is similar, irrespective of whether it is bound to a homo- or a heteroduplex. The results envisage a minimal model of three conformational states of MutS as reflected in: (i) a specific and highly stable MutS–mismatch complex in the absence of a nucleotide; (ii) a specific but less stable complex in the presence of ATP hydrolysis; and (iii) an irreversibly dissociated complex in the presence of ATP binding (ATPγS). Such transitions are of relevance to the protein’s function in vivo where it has to first recognize a mismatch, followed by a search for hemimethylated sites.     

Functional Analyses of Escherichia coli MutS-β Clamp Interaction In Vitro and In Vivo

Escherichia coli MutS is a highly conserved mismatch repair (MMR) protein that plays a key role in recognizing DNA mismatches and the early steps of MMR. Previous studies revealed an interaction between MutS and the replicative protein β clamp, but it remains unclear whether the interaction functions during the process of MMR. In order to provide insight into the significance of this interaction, Far Western, Surface plasmon resonance and cell survival/mutagenesis assays were used to determine its possible influences on the in vitro and in vivo properties of MutS. The results show that a quintuple mutation of MutS residues 812–816 (MutS^βC), or single alanine substitution mutation of MutS residues M813 or L815 completely blocks binding of MutS to β clamp. Wild type β clamp interferes with DNA binding by MutS. When treated with the base analog 2-aminopurine, MutS^βC confers more mutations and less cellular growth rate in the mutS-deficient strain than the wild type MutS. These data indicate that the MutS-β interaction has functional consequences during MMR in E. coli.     

Slow Conformational Changes in MutS and DNA Direct Ordered Transitions between Mismatch Search,Recognition and Signaling of DNA Repair

MutS functions in mismatch repair (MMR) to scan DNA for errors, identify a target site and trigger subsequent events in the pathway leading to error removal and DNA re-synthesis. These actions, enabled by the ATPase activity of MutS, are now beginning to be analyzed from the perspective of the protein itself. This study provides the first ensemble transient kinetic data on MutS conformational dynamics as it works with DNA and ATP in MMR. Using a combination of fluorescence probes (on Thermus aquaticus MutS and DNA) and signals (intensity, anisotropy and resonance energy transfer), we have monitored the timing of key conformational changes in MutS that are coupled to mismatch binding and recognition, ATP binding and hydrolysis, as well as sliding clamp formation and signaling of repair. Significant findings include (a) a slow step that follows weak initial interaction between MutS and DNA, in which concerted conformational changes in both macromolecules control mismatch recognition, and (b) rapid, binary switching of MutS conformations that is concerted with ATP binding and hydrolysis and (c) is stalled after mismatch recognition to control formation of the ATP-bound MutS sliding clamp. These rate-limiting pre- and post-mismatch recognition events outline the mechanism of action of MutS on DNA during initiation of MMR.     

Activity of E. coli ClpA bound by nucleoside diphosphates and triphosphates

The Escherichia coli ClpA protein is a molecular chaperone that binds and translocates protein substrates into the proteolytic cavity of the tetradecameric serine protease ClpP. In the absence of ClpP, ClpA can remodel protein complexes. In order for ClpA to bind protein substrates targeted for removal or remodeling, ClpA requires nucleoside triphosphate binding to first assemble into a hexamer. Here we report the assembly properties of ClpA in the presence of the nucleoside diphosphates and triphosphates ADP, adenosine 5′-[γ-thio]triphosphate, adenosine 5′-(β,γ-imido)triphosphate, β,γ-methyleneadenosine 5′-triphosphate, and adenosine diphosphate beryllium fluoride. In addition to examining the assembly of ClpA in the presence of various nucleotides and nucleotide analogues, we have also correlated the assembly state of ClpA in the presence of these nucleotides with both polypeptide binding activity and enzymatic activity, specifically ClpA-catalyzed polypeptide translocation. Here we show that all of the selected nucleotides, including ADP, promote the assembly of ClpA. However, only adenosine 5′-[γ-thio]triphosphate and adenosine 5′-(β,γ-imido)triphosphate promote the formation of an oligomer of ClpA that is active in polypeptide binding and translocation. These results suggest that the presence of γ phosphate may serve to switch ClpA into a conformational state with high peptide binding activity, whereas affinity is severely attenuated when ADP is bound.     

One tube mutation detection using sensitive fluorescent dyeing of MutS protected DNA

A novel, universal method for mutation detection utilising the ability of MutS protein to recognise DNA incomplementarities is proposed. The examined and reference DNA fragments are PCR amplified. The PCR products are purified, mixed, heated and cooled to form heteroduplexes. In the case of mutation the heteroduplex DNA containing mismatch is protected against exonuclease digestion by MutS, while the DNA without mismatches is degraded. The protection effect is visualised by the direct addition of a highly sensitive fluorescent dye (SYBR-Gold) selectively binding DNA. The Thermus thermophilus recombined His-tagged MutS and 3′–5′ exonuclease activity of T4 DNA polymerase were used in the assay.     

Sequence-specific and DNA structure-dependent interactions of Escherichia coli MutS and human p53 with DNA

Many proteins involved in DNA repair systems interact with DNA that has structure altered from the typical B-form helix. Using magnetic beads to immobilize DNAs containing various types of structures, we evaluated the in vitro binding activities of two well-characterized DNA repair proteins, Escherichia coli MutS and human p53. E. coli MutS bound to double-stranded DNAs, with higher affinity for a G/T mismatch compared to a G/A mismatch and highest affinity for larger non-B-DNA structures. E. coli MutS bound best to DNA between pH 6 and 9. Experiments discriminated between modes of p53–DNA binding, and increasing ionic strength reduced p53 binding to nonspecific double-stranded DNA, but had minor effects on binding to consensus response sequences or single-stranded DNA. Compared to nonspecific DNA sequences, p53 bound with a higher affinity to mismatches and base insertions, while binding to various hairpin structures was similar to that observed to its consensus DNA sequence. For hairpins containing CTG repeats, the extent of p53 binding was proportional to the size of the repeat. In summary, using the flexibility of the magnetic bead separation assay we demonstrate that pH and ionic strength influence the binding of two DNA repair proteins to a variety of DNA structures.     

Identification of Critical Residues for the Tight Binding of Both Correct and Incorrect Nucleotides to Human DNA Polymerase λ

DNA polymerase λ (Pol λ) is a novel X-family DNA polymerase that shares 34% sequence identity with DNA polymerase β. Pre-steady-state kinetic studies have shown that the Pol λ-DNA complex binds both correct and incorrect nucleotides 130-fold tighter, on average, than the DNA polymerase β-DNA complex, although the base substitution fidelity of both polymerases is 10^− 4 to 10^− 5. To better understand Pol λ's tight nucleotide binding affinity, we created single-substitution and double-substitution mutants of Pol λ to disrupt the interactions between active-site residues and an incoming nucleotide or a template base. Single-turnover kinetic assays showed that Pol λ binds to an incoming nucleotide via cooperative interactions with active-site residues (R386, R420, K422, Y505, F506, A510, and R514). Disrupting protein interactions with an incoming correct or incorrect nucleotide impacted binding to each of the common structural moieties in the following order: triphosphate ? base > ribose. In addition, the loss of Watson-Crick hydrogen bonding between the nucleotide and the template base led to a moderate increase in K_d. The fidelity of Pol λ was maintained predominantly by a single residue, R517, which has minor groove interactions with the DNA template.     

Is Thymidine Glycol Containing DNA a Substrate of E. coli DNA Mismatch Repair System?

The DNA mismatch repair (MMR) system plays a crucial role in the prevention of replication errors and in the correction of some oxidative damages of DNA bases. In the present work the most abundant oxidized pyrimidine lesion, 5,6-dihydro-5,6-dihydroxythymidine (thymidine glycol, Tg) was tested for being recognized and processed by the E. coli MMR system, namely complex of MutS, MutL and MutH proteins. In a partially reconstituted MMR system with MutS-MutL-MutH proteins, G/Tg and A/Tg containing plasmids failed to provoke the incision of DNA. Tg residue in the 30-mer DNA duplex destabilized double helix due to stacking disruption with neighboring bases. However, such local structural changes are not important for E. coli MMR system to recognize this lesion. A lack of repair of Tg containing DNA could be due to a failure of MutS (a first acting protein of MMR system) to interact with modified DNA in a proper way. It was shown that Tg in DNA does not affect on ATPase activity of MutS. On the other hand, MutS binding affinities to DNA containing Tg in G/Tg and A/Tg pairs are lower than to DNA with a G/T mismatch and similar to canonical DNA. Peculiarities of MutS interaction with DNA was monitored by Förster resonance energy transfer (FRET) and fluorescence anisotropy. Binding of MutS to Tg containing DNAs did not result in the formation of characteristic DNA kink. Nevertheless, MutS homodimer orientation on Tg-DNA is similar to that in the case of G/T-DNA. In contrast to G/T-DNA, neither G/Tg- nor A/Tg-DNA was able to stimulate ADP release from MutS better than canonical DNA. Thus, Tg residue in DNA is unlikely to be recognized or processed by the E. coli MMR system. Probably, the MutS transformation to active “sliding clamp” conformation on Tg-DNA is problematic.     

Multiple global conformational states of the hexameric RepA helicase of plasmid RSF1010 with different ssDNA-binding capabilities are induced by different numbers of bound nucleotides. Analytical ultracentrifugation and dynamic light scattering studies

Global conformational transitions of the hexameric RepA helicase of plasmid RSF1010, induced by the nucleoside tri and di-phosphate binding, have been examined using analytical ultracentrifugation and dynamic light scattering techniques. The global structure of the RepA hexamer in solution, modeled as an oblate ellipsoid of revolution, is very different from its crystal structure, with the axial ratio of the ellipsoid being ∼4.5 as compared to only ∼2.4 in the crystal structure. The large axial ratio and the experimentally determined partial specific volume strongly suggest that, in solution, the diameter of the cross-channel of the hexamer is larger than ∼17 Å seen in the crystal. The global conformation of the helicase is modulated by a specific number of bound nucleotides. The enzyme exists in at least four conformational states, occurring sequentially as a function of the number of bound cofactors. These conformational states are different for ADP, as compared to β,γ-imidoadenosine 5′-triphosphate (AMP-PNP). Modulation of the global structure is separated into two phases, different for complexes with up to three bound nucleotides, from the effect observed at the saturating level of cofactors. This heterogeneity indicates different functional roles of the two modulation processes. Nucleotide control of helicase - single-stranded (ss)DNA interactions occurs through affecting the enzyme structure and the ssDNA affinity prior to DNA binding. Only one conformational state of the helicase, with two AMP-PNP molecules bound, has dramatically higher ssDNA-affinities than the complexes with ADP. Moreover the same state also has an increased site-size of the enzyme - ssDNA complexes. The implications of these findings for functional activities of a hexameric helicase are discussed.     

Kinetic control of Mg2+-dependent melting of duplex DNA ends by Escherichia coli RecBC

Escherichia coli RecBCD is a highly processive DNA helicase involved in double-strand break repair and recombination that possesses two helicase/translocase subunits with opposite translocation directionality (RecB (3′ to 5′) and RecD (5′ to 3′)). RecBCD has been shown to melt out ∼ 5-6 bp upon binding to a blunt-ended duplex DNA in a Mg²⁺-dependent, but ATP-independent reaction. Here, we examine the binding of E. coli RecBC helicase (minus RecD), also a processive helicase, to duplex DNA ends in the presence and in the absence of Mg²⁺ in order to determine if RecBC can also melt a duplex DNA end in the absence of ATP. Equilibrium binding of RecBC to DNA substrates with ends possessing pre-formed 3′ and/or 5′ single-stranded (ss)-(dT)_n flanking regions (tails) (n ranging from zero to 20 nt) was examined by competition with a fluorescently labeled reference DNA and by isothermal titration calorimetry. The presence of Mg²⁺ enhances the affinity of RecBC for DNA ends possessing 3′ or 5′-(dT)_n ssDNA tails with n < 6 nt, with the relative enhancement decreasing as n increases from zero to six nt. No effect of Mg²⁺ was observed for either the binding constant or the enthalpy of binding (ΔH_obs) for RecBC binding to DNA with ssDNA tail lengths, n ≥ 6 nucleotides. Upon RecBC binding to a blunt duplex DNA end in the presence of Mg²⁺, at least 4 bp at the duplex end become accessible to KMnO₄ attack, consistent with melting of the duplex end. Since Mg²⁺ has no effect on the affinity or binding enthalpy of RecBC for a DNA end that is fully pre-melted, this suggests that the role of Mg²⁺ is to overcome a kinetic barrier to melting of the DNA by RecBC and presumably also by RecBCD. These data also provide an accurate estimate (ΔH_obs = 8 ± 1 kcal/mol) for the average enthalpy change associated with the melting of a DNA base-pair by RecBC.     

Modulation of MutS ATP-dependent functional activities by DNA containing a cisplatin compound lesion (base damage and mismatch)

DNA damage-dependent signaling by the DNA mismatch repair (MMR) system is thought to mediate cytotoxicity of the anti-tumor drug cisplatin through molecular mechanisms that could differ from those required for normal mismatch repair. The present study investigated whether ATP-dependent biochemical properties of Escherichia coli MutS protein differ when the protein interacts with a DNA oligonucleotide containing a GT mismatch versus a unique site specifically placed cisplatin compound lesion, a cisplatin 1,2-d(GpG) intrastrand cross-link with a mispaired thymine opposite the 3' platinated guanine. MutS exhibited substantial affinity for this compound lesion in hydrolytic and in non-hydrolytic conditions of ATP, contrasting with the normal nucleotide inhibition effect of mispair binding. The cisplatin compound lesion was also shown to stimulate poorly MutS ATPase activity to approach the hydrolysis rate induced by nonspecific DNA. Moreover, MutS undergoes distinct conformation changes in the presence of the compound lesion and ATP under hydrolytic conditions as shown by limited proteolysis. In the absence of MutS, the cisplatin compound lesion was shown to induce a 39 degrees rigid bending of the DNA double helix contrasting with an unbent state for DNA containing a GT mispair. Furthermore, an unbent DNA substrate containing a monofunctional adduct mimicking a cisplatin residue failed to form a persistent nucleoprotein complex with MutS in the presence of adenine nucleotide. We propose that DNA bending could play a role in MutS biochemical modulations induced by a compound lesion and that cisplatin DNA damage signaling by the MMR system could be modulated in a direct mode.     

Characterisation of the Nucleotide Exchange Factor ITSN1L: Evidence for a Kinetic Discrimination of GEF-Stimulated Nucleotide Release from Cdc42

Cdc42, a member of the Ras superfamily of small guanine nucleotide binding proteins, plays an important role in regulating the actin cytoskeleton, intracellular trafficking, and cell polarity. Its activation is controlled by guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of bound guanosine-5′-diphosphate (GDP) to allow guanosine-5′-triphosphate (GTP) binding. Here, we investigate the exchange factor activity of the Dbl-homology domain containing constructs of the adaptor protein Intersectin1L (ITSN1L), which is a specific GEF for Cdc42. A detailed kinetic characterisation comparing ITSN1L-mediated nucleotide exchange on Cdc42 in its GTP- versus GDP-bound state reveals a kinetic discrimination for GEF-stimulated dissociation of GTP: The maximum acceleration of the intrinsic mGDP [2′/3′-O-(N-methyl-anthraniloyl)-GDP] release from Cdc42 by ITSN1L is accelerated at least 68,000-fold, whereas the exchange of mGTP [2′/3′-O-(N-methyl-anthraniloyl)-GTP] is stimulated only up to 6000-fold at the same GEF concentration. The selectivity in nucleotide exchange kinetics for GDP over GTP is even more pronounced when a Cdc42 mutant, F28L, is used, which is characterised by fast intrinsic dissociation of nucleotides. We furthermore show that both GTP and Mg²⁺ ions are required for the interaction with effectors. We suggest a novel model for selective nucleotide exchange residing on a conformational change of Cdc42 upon binding of GTP, which enables effector binding to the Cdc42 · GTP complex but, at the same time, excludes efficient modulation by the GEF. The higher exchange activity of ITSN1L towards the GDP-bound conformation of Cdc42 could represent an evolutionary adaptation of this GEF that ensures nucleotide exchange towards the formation of the signalling-active GTP-bound form of Cdc42 and avoids dissociation of the active complex.     

Distinct Requirements for 5′-Monophosphate-assisted RNA Cleavage by Escherichia coli RNase E and RNase G

RNase E and RNase G are homologous endonucleases that play important roles in RNA processing and decay in Escherichia coli and related bacterial species. Rapid mRNA degradation is facilitated by the preference of both enzymes for decay intermediates whose 5′ end is monophosphorylated. In this report we identify key characteristics of RNA that influence the rate of 5′-monophosphate-assisted cleavage by these two ribonucleases. In vitro, both require at least two and prefer three or more unpaired 5′-terminal nucleotides for such cleavage; however, RNase G is impeded more than RNase E when fewer than four unpaired nucleotides are present at the 5′ end. Each can tolerate any unpaired nucleotide (A, G, C, or U) at either of the first two positions, with only modest biases. The optimal spacing between the 5′ end and the scissile phosphate appears to be eight nucleotides for RNase E but only six for RNase G. 5′-Monophosphate-assisted cleavage also occurs, albeit more slowly, when that spacing is greater or at most one nucleotide shorter than the optimum, but there is no simple inverse relationship between increased spacing and the rate of cleavage. These properties are also manifested during 5′-end-dependent mRNA degradation in E. coli.