Distinct MutS DNA-binding Modes That Are Differentially Modulated by ATP Binding and Hydrolysis

Blackwell, Leonard J.; Bjornson, Keith P.; Allen, Dwayne J.; Modrich, Paul

doi:10.1074/jbc.m104256200

Cited by 89 publications

(114 citation statements)

References 32 publications

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“…DNA binding isotherms for MutS and MutS E694A were determined by titration of chip-bound DNA with increasing concentrations of MutS or MutS E694A. Maximum binding values obtained in this manner were analyzed as a function of protein concentration as described previously (30). We have previously found that isolated MutS is free of ATP but can contain variable, substoichiometric quantities of bound ADP (Ͻ0.01-0.15 mol/mol of monomer) (36).…”

Section: Methodsmentioning

confidence: 99%

“…2 Recently modified purification methods (Ref. 30 and "Experimental Procedures") typically yield MutS isolates that are free of detectable nucleotide. Preparations isolated as described under "Experimental Procedures" contained Ͻ0.01 mol of adenine nucleotide per mol of MutS monomer.…”

Section: Methodsmentioning

confidence: 99%

“…These two pools were separately subjected to chromatography on Heparin Hi-Trap columns as described above. Forty-one and 201-bp G-T heteroduplexes and otherwise identical A⅐T homoduplexes tagged with 5Ј-terminal biotin at one end were prepared as described previously (27,30).…”

Section: Methodsmentioning

confidence: 99%

“…coli MutS and MutS E694A were purified as described (30) except that chromatography on a heparin affinity column was added as a last step. Pooled fractions eluting from the hydroxylapatite column (75-150 mg, ϳ0.5 mg/ml) were dialyzed against 20 mM KPO 4 , pH 7.4, 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol and loaded onto a Heparin Hi-Trap column (Amersham Biosciences, 5 ml) equilibrated with 20 mM KPO 4 , pH 7.4, 100 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol.…”

Section: Methodsmentioning

confidence: 99%

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Hydrolytically Deficient MutS E694A Is Defective in the MutL-dependent Activation of MutH and in the Mismatch-dependent Assembly of the MutS · MutL · Heteroduplex Complex

Baitinger

Burdett

Modrich

2003

Journal of Biological Chemistry

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Mismatch recognition by MutS initiates mismatch repair in bacterial cells (1-3). MutS recruits MutL to the heteroduplex in an ATP-dependent fashion (4 -6), and assembly of the MutL⅐MutS⅐heteroduplex ternary complex is sufficient to activate the MutH endonuclease, which incises the unmethylated strand at a hemimethylated d(GATC) strand signal (7). The ensuing strand break, which may reside either 3Ј or 5Ј to the mismatch, serves as the site for initiation of excision by a system comprised of DNA helicase II and an appropriate 3Ј to 5Ј or 5Ј to 3Ј single-strand specific exonuclease (8 -13). This bidirectional excision capability implies that the mismatch repair system must establish the relative orientation of the mismatch and strand break that directs the reaction. This is necessary to ensure loading of a 3Ј to 5Ј excision system when the nick resides 3Ј to the mispair and a 5Ј to 3Ј system when it is located 5Ј to the mismatch.In addition to its mismatch binding site, MutS has a carboxyl-terminal ATPase that is required for function of the protein in mismatch repair (7,14,15), and this element is conserved in MutS homologs in higher cells (16 -20). Structural studies have shown that MutS is a member of the ABC (adenine nucleotide binding cassette) family (21-23), which is largely comprised of proteins that couple the energy of ATP hydrolysis to transport of molecules across biological membranes (24,25).Several models for ATPase function in MutS homolog action have been suggested. One class of mechanism is based on a variety of observations indicating that in the presence of ATP, MutS homologs can leave a mismatch by movement along the helix (26 -29). This movement is postulated to link mismatch recognition to activation of downstream events at the strand break that directs excision, and can in principle account for the orientation-dependent loading of the excision system at the strand break that is necessitated by the bidirectional nature of the repair system. Two types of mechanisms have been proposed to explain ATP-dependent movement of MutS homologs along the DNA contour. One model posits that movement depends on ATP hydrolysis by the DNA-bound protein. The alternate molecular switch model postulates a G-protein like mechanism whereby binding of a MutS⅐ADP complex to a mismatch promotes ATP exchange for ADP, with the resulting MutS⅐ATP complex diffusing freely along the helix. Evidence consistent with both the hydrolytic model (26, 27, 29 -31) and the molecular switch model (28, 32) is available.A distinct role for ATP binding and hydrolysis has been proposed based on use of a trans assay for MutH activation (23). In this work, a mismatch on one oligonucleotide duplex was shown to lead to MutH activation and d(GATC) incision on a second synthetic duplex in a reaction dependent on MutL and MutS. These observations have led to the suggestion that once mismatch recognition occurs, MutS remains bound to the mispair during the course of repair, with mismatch-strand signal interaction mediated by DNA bending (23)....

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Hydrolytically Deficient MutS E694A Is Defective in the MutL-dependent Activation of MutH and in the Mismatch-dependent Assembly of the MutS · MutL · Heteroduplex Complex

Baitinger

Burdett

Modrich

2003

Journal of Biological Chemistry

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“…ATP binding and hydrolysis appear to modulate the interactions between MutS/Msh and DNA as well as other proteins in the repair pathway; thus, understanding how MutS/Msh proteins utilize ATP is necessary for understanding how they function in DNA mismatch repair. Several model mechanisms have been proposed for MutS/Msh action upon mismatch recognition: (a) MutS/Msh proteins translocate on DNA, fuelled by ATP binding and hydrolysis, possibly to interact with other proteins on DNA and coordinate mismatch recognition with downstream events such as initiation of strand excision and DNA resynthesis [8][9][10]; (b) upon binding ATP MutS/Msh proteins form sliding clamps that diffuse freely on DNA, again, to contact downstream repair proteins and direct repair [11,12]; (c) MutS/Msh proteins utilize ATP binding and hydrolysis to modulate their interaction with DNA, while remaining at the mismatch to direct repair [13][14][15][16][17]. At present, experimental data are available in support of each of these very different model mechanisms, therefore the investigation into MutS/Msh DNA binding and ATPase activities continues.…”

Section: Introductionmentioning

confidence: 99%

Contribution of Msh2 and Msh6 subunits to the asymmetric ATPase and DNA mismatch binding activities of Saccharomyces cerevisiae Msh2–Msh6 mismatch repair protein

et al. 2006

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Previous analyses of both Thermus aquaticus MutS homodimer and Saccharomyces cerevisiae Msh2-Msh6 heterodimer have revealed that the subunits in these protein complexes bind and hydrolyze ATP asymmetrically, emulating their asymmetric DNA binding properties. In the MutS homodimer, one subunit (S 1 ) binds ATP with high affinity and hydrolyzes it rapidly, while the other subunit (S 2 ) binds ATP with lower affinity and hydrolyzes it at an apparently slower rate. Interaction of MutS with mismatched DNA results in suppression of ATP hydrolysis at S 1 -but which of these subunits, S 1 or S 2 , makes specific contact with the mismatch (e.g., base stacking by a conserved phenylalanine residue) remains unknown. In order to answer this question and to clarify the links between the DNA binding and ATPase activities of each subunit in the dimer, we made mutations in the ATPase sites of Msh2 and Msh6 and assessed their impact on the activity of the Msh2-Msh6 heterodimer (in Msh2-Msh6, only Msh6 makes base specific contact with the mismatch). The key findings are: (a) Msh6 hydrolyzes ATP rapidly, and thus resembles the S 1 subunit of the MutS homodimer, (b) Msh2 hydrolyzes ATP at a slower rate, and thus resembles the S 2 subunit of MutS, (c) though itself an apparently weak ATPase, Msh2 has a strong influence on the ATPase activity of Msh6, (d) Msh6 binding to mismatched DNA results in suppression of rapid ATP hydrolysis, revealing a "cis" linkage between its mismatch recognition and ATPase activities, (e) the resultant Msh2-Msh6 complex, with both subunits in the ATP-bound state, exhibits altered interactions with the mismatch.

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Chemie und Biologie der DNA‐Reparatur

Schärer

2003

Angewandte Chemie

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Zahlreiche Agentien endogenen und exogenen Ursprungs schädigen die DNA in unserem Genom. Es existieren unterschiedliche Reparatursysteme, die Schäden in der DNA erkennen und durch eine Vielzahl von Reaktionssequenzen beheben können. Defekte DNA‐Reparaturproteine hängen mit einigen erblich bedingten Syndromen zusammen, die eine Prädisposition für Krebs aufweisen. Während die DNA‐Reparatur einerseits essenziell für eine gesunde Zelle ist, beeinträchtigen DNA‐Reparaturenzyme andererseits die Effizienz vieler Antitumorwirkstoffe, deren Wirkung auf der Schädigung von DNA beruht, sodass DNA‐Reparaturenzyme auch hinsichtlich des Wirkstoff‐Designs von großer Bedeutung sind. DNA‐Reparaturprozesse variieren stark in ihrer Art und Komplexität. Während in einem Fall nur ein einziges Enzym benötigt wird, ist an anderen Pfaden ein koordiniertes Zusammenspiel von dreißig oder mehr Proteinen beteiligt. Unser Kenntnisstand der genetischen, biochemischen und strukturellen Grundlagen der DNA‐Reparatur und damit verwandter Prozesse hat sich in den letzten Jahren stark verbessert. Dieser Aufsatz fasst die jüngsten Forschungsergebnisse auf diesem Gebiet zusammen.

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Distinct MutS DNA-binding Modes That Are Differentially Modulated by ATP Binding and Hydrolysis

Cited by 89 publications

References 32 publications

Hydrolytically Deficient MutS E694A Is Defective in the MutL-dependent Activation of MutH and in the Mismatch-dependent Assembly of the MutS · MutL · Heteroduplex Complex

Hydrolytically Deficient MutS E694A Is Defective in the MutL-dependent Activation of MutH and in the Mismatch-dependent Assembly of the MutS · MutL · Heteroduplex Complex

Contribution of Msh2 and Msh6 subunits to the asymmetric ATPase and DNA mismatch binding activities of Saccharomyces cerevisiae Msh2–Msh6 mismatch repair protein

Chemie und Biologie der DNA‐Reparatur

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