scholarly journals The properties of Msh2–Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair

2018 ◽  
Vol 293 (47) ◽  
pp. 18055-18070 ◽  
Author(s):  
William J. Graham ◽  
Christopher D. Putnam ◽  
Richard D. Kolodner
Keyword(s):  
Mismatch Repair ◽  
Binding Sites ◽  
Signal Amplification ◽  
Dna Mismatch Repair ◽  
Nucleotide Binding ◽  
Atp Binding ◽  
Dna Mismatch ◽  
Nucleotide Binding Sites ◽  
Sliding Clamp ◽  
Atp Concentration

DNA mismatch repair (MMR) corrects mispaired DNA bases and small insertion/deletion loops generated by DNA replication errors. After binding a mispair, the eukaryotic mispair recognition complex Msh2–Msh6 binds ATP in both of its nucleotide-binding sites, which induces a conformational change resulting in the formation of an Msh2–Msh6 sliding clamp that releases from the mispair and slides freely along the DNA. However, the roles that Msh2–Msh6 sliding clamps play in MMR remain poorly understood. Here, using Saccharomyces cerevisiae, we created Msh2 and Msh6 Walker A nucleotide–binding site mutants that have defects in ATP binding in one or both nucleotide-binding sites of the Msh2–Msh6 heterodimer. We found that these mutations cause a complete MMR defect in vivo. The mutant Msh2–Msh6 complexes exhibited normal mispair recognition and were proficient at recruiting the MMR endonuclease Mlh1–Pms1 to mispaired DNA. At physiological (2.5 mm) ATP concentration, the mutant complexes displayed modest partial defects in supporting MMR in reconstituted Mlh1–Pms1-independent and Mlh1–Pms1-dependent MMR reactions in vitro and in activation of the Mlh1–Pms1 endonuclease and showed a more severe defect at low (0.1 mm) ATP concentration. In contrast, five of the mutants were completely defective and one was mostly defective for sliding clamp formation at high and low ATP concentrations. These findings suggest that mispair-dependent sliding clamp formation triggers binding of additional Msh2–Msh6 complexes and that further recruitment of additional downstream MMR proteins is required for signal amplification of mispair binding during MMR.

scholarly journals Cryo-EM structures reveal how ATP and DNA binding in MutS coordinate the sequential steps of DNA mismatch repair

2021 ◽  
Author(s):  
Alessandro Borsellini ◽  
Vladislav Kunetsky ◽  
Peter Friedhoff ◽  
Meindert H. Lamers
Keyword(s):  
Dna Binding ◽  
Mismatch Repair ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Dna Mismatch Repair ◽  
Atp Binding ◽  
Dna Mismatch ◽  
Sliding Clamp ◽  
Adp Release ◽  
Atp Binding And Hydrolysis

DNA mismatch repair detects and removes mismatches from DNA reducing the error rate of DNA replication a 100-1000 fold. The MutS protein is one of the key players that scans for mismatches and coordinates the repair cascade. During this, MutS undergoes multiple conformational changes that initiate the subsequent steps, in response to ATP binding, hydrolysis, and release. How ATP induces the different conformations in MutS is not well understood. Here we present four cryo-EM structures of Escherichia coli MutS at sequential stages of the ATP hydrolysis cycle. These structures reveal how ATP binding and hydrolysis induces a closing and opening of the MutS dimer, respectively. Additional biophysical analysis furthermore explains how DNA binding modulates the ATPase cycle by preventing hydrolysis during scanning and mismatch binding, while preventing ADP release in the sliding clamp state. Nucleotide release is achieved when MutS encounters single stranded DNA that is produced during the removal of the daughter strand. This way, the combination of the ATP binding and hydrolysis and its modulation by DNA enable MutS to adopt different conformations needed to coordinate the sequential steps of the mismatch repair cascade.

scholarly journals Functional Studies on the Candidate ATPase Domains of Saccharomyces cerevisiae MutLα

2000 ◽  
Vol 20 (17) ◽  
pp. 6390-6398 ◽  
Author(s):  
Phuoc T. Tran ◽  
R. Michael Liskay
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mismatch Repair ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Dna Mismatch Repair ◽  
Limited Proteolysis ◽  
Atp Binding ◽  
Dna Mismatch ◽  
Functional Studies

ABSTRACT Saccharomyces cerevisiae MutL homologues Mlh1p and Pms1p form a heterodimer, termed MutLα, that is required for DNA mismatch repair after mismatch binding by MutS homologues. Recent sequence and structural studies have placed the NH2 termini of MutL homologues in a new family of ATPases. To address the functional significance of this putative ATPase activity in MutLα, we mutated conserved motifs for ATP hydrolysis and ATP binding in both Mlh1p and Pms1p and found that these changes disrupted DNA mismatch repair in vivo. Limited proteolysis with purified recombinant MutLα demonstrated that the NH2 terminus of MutLα undergoes conformational changes in the presence of ATP and nonhydrolyzable ATP analogs. Furthermore, two-hybrid analysis suggested that these ATP-binding-induced conformational changes promote an interaction between the NH2 termini of Mlh1p and Pms1p. Surprisingly, analysis of specific mutants suggested differential requirements for the ATPase motifs of Mlh1p and Pms1p during DNA mismatch repair. Taken together, these results suggest that MutLα undergoes ATP-dependent conformational changes that may serve to coordinate downstream events during yeast DNA mismatch repair.

Discrete mutations introduced in the predicted nucleotide-binding sites of the mdr1 gene abolish its ability to confer multidrug resistance

1989 ◽  
Vol 9 (12) ◽  
pp. 5289-5297
Author(s):  
M Azzaria ◽  
E Schurr ◽  
P Gros
Keyword(s):  
Drug Resistance ◽  
Biological Activity ◽  
Multidrug Resistance ◽  
Amino Acid ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Nucleotide Binding ◽  
Atp Binding ◽  
Nucleotide Binding Sites ◽  
The Core

In cells stably transfected and overexpressing the mouse mdr1 gene, multidrug resistance is associated with an increased ATP-dependent drug efflux. Analysis of the predicted amino acid sequence of the MDR1 protein revealed the presence of two putative nucleotide-binding sites (NBS). To assess the functional importance of these NBS in the overall drug resistance phenotype conferred by mdr1, we introduced amino acid substitutions in the core consensus sequence for nucleotide binding, GXGKST. Mutants bearing the sequence GXAKST or GXGRST at either of the two NBS of mdr1 and a double mutant harboring the sequence GXGRST at both NBS were generated. The integrity of the two NBS was essential for the biological activity of mdr1, since all five mutants were unable to confer drug resistance to hamster drug-sensitive cells in transfection experiments. Conversely, a lysine-to-arginine substitution outside the core consensus sequence had no effect on the activity of mdr1. Failure to reduce intracellular accumulation of [3H]vinblastine paralleled the loss of activity in cell clones expressing mutant MDR1 proteins. However, the ability to bind the photoactivatable ATP analog 8-azido ATP was retained in the five inactive MDR1 mutants. This result implies that an essential step subsequent to ATP binding is impaired in these mutants, possibly ATP hydrolysis or secondary conformational changes induced by ATP-binding or hydrolysis. Our results suggest that the two NBS function in a cooperative fashion, since mutations in a single NBS completely abrogated the biological activity of mdr1.

scholarly journals Dependence on Membrane Lipids of the Effect of Vanadate on Calcium and ATP Binding to Sarcoplasmic Reticulum ATPase

1984 ◽  
Vol 39 (11-12) ◽  
pp. 1137-1140 ◽  
Author(s):  
Pankaj Medda ◽  
Wilhelm Hasselbach
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Binding Sites ◽  
Calcium Binding ◽  
Membrane Lipids ◽  
Nucleotide Binding ◽  
Atp Binding ◽  
Transport Atpase ◽  
Nucleotide Binding Sites ◽  
High Affinity ◽  
Calcium Binding Sites

Abstract The affinity of the sarcoplasmic reticulum transport ATPase for calcium and ATP is not affected by lipid depriviation while vanadate binding is completely abolished. Lipid substitution restores vanadate binding as well as the vanadate induced disappearance of the enzyme’s high affinity calcium and nucleotide binding sites. Nucleotide binding is simultaneously restored with the displacement of vanadate from the enzyme following the occupation of its low affinity calcium binding sites.

scholarly journals Adenosine nucleotide binding sites on Complex V from diverse organisms

2021 ◽  
Author(s):  
Abhinav Parashar ◽  
Kelath Murali Manoj
Keyword(s):  
Binding Sites ◽  
In Silico ◽  
Nucleotide Binding ◽  
Atp Binding ◽  
Atp Binding Site ◽  
In Silico Docking ◽  
Epsilon Subunit ◽  
Nucleotide Binding Sites ◽  
Alpha Beta ◽  
Adenosine Nucleotide

Using in silico docking approaches, we scan the various subunits of Complex V (FoF1ATPase) for putative adenosine nucleotide binding sites. We find that multiple generic ADP/ATP binding sites are present on the alpha-beta binding sites and a conserved ATP binding site is present on the epsilon subunit. These findings support the murburn model of Complex V.

scholarly journals MutS/MutL crystal structure reveals that the MutS sliding clamp loads MutL onto DNA

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Flora S Groothuizen ◽  
Ines Winkler ◽  
Michele Cristóvão ◽  
Alexander Fish ◽  
Herrie HK Winterwerp ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Dna Replication ◽  
Mismatch Repair ◽  
Conformational Changes ◽  
Dna Mismatch Repair ◽  
Transient State ◽  
Dna Mismatch ◽  
Sliding Clamp ◽  
Downstream Effectors ◽  
A Site

To avoid mutations in the genome, DNA replication is generally followed by DNA mismatch repair (MMR). MMR starts when a MutS homolog recognizes a mismatch and undergoes an ATP-dependent transformation to an elusive sliding clamp state. How this transient state promotes MutL homolog recruitment and activation of repair is unclear. Here we present a crystal structure of the MutS/MutL complex using a site-specifically crosslinked complex and examine how large conformational changes lead to activation of MutL. The structure captures MutS in the sliding clamp conformation, where tilting of the MutS subunits across each other pushes DNA into a new channel, and reorientation of the connector domain creates an interface for MutL with both MutS subunits. Our work explains how the sliding clamp promotes loading of MutL onto DNA, to activate downstream effectors. We thus elucidate a crucial mechanism that ensures that MMR is initiated only after detection of a DNA mismatch.

scholarly journals A Lynch syndrome-associated mutation at a Bergerat ATP-binding fold destabilizes the structure of the DNA mismatch repair endonuclease MutL

2020 ◽  
Vol 295 (33) ◽  
pp. 11643-11655
Author(s):  
Keisuke Izuhara ◽  
Kenji Fukui ◽  
Takeshi Murakawa ◽  
Seiki Baba ◽  
Takashi Kumasaka ◽  
...  
Keyword(s):  
Dna Binding ◽  
Lynch Syndrome ◽  
Atpase Activity ◽  
Mismatch Repair ◽  
Dna Mismatch Repair ◽  
Limited Proteolysis ◽  
Atp Binding ◽  
Aquifex Aeolicus ◽  
Dna Mismatch ◽  
Genes Encoding

In humans, mutations in genes encoding homologs of the DNA mismatch repair endonuclease MutL cause a hereditary cancer that is known as Lynch syndrome. Here, we determined the crystal structures of the N-terminal domain (NTD) of MutL from the thermophilic eubacterium Aquifex aeolicus (aqMutL) complexed with ATP analogs at 1.69–1.73 Å. The structures revealed significant structural similarities to those of a human MutL homolog, postmeiotic segregation increased 2 (PMS2). We introduced five Lynch syndrome-associated mutations clinically found in human PMS2 into the aqMutL NTD and investigated the protein stability, ATPase activity, and DNA-binding ability of these protein variants. Among the mutations studied, the most unexpected results were obtained for the residue Ser34. Ser34 (Ser46 in PMS2) is located at a previously identified Bergerat ATP-binding fold. We found that the S34I aqMutL NTD retains ATPase and DNA-binding activities. Interestingly, CD spectrometry and trypsin-limited proteolysis indicated the disruption of a secondary structure element of the S34I NTD, destabilizing the overall structure of the aqMutL NTD. In agreement with this, the recombinant human PMS2 S46I NTD was easily digested in the host Escherichia coli cells. Moreover, other mutations resulted in reduced DNA-binding or ATPase activity. In summary, using the thermostable aqMutL protein as a model molecule, we have experimentally determined the effects of the mutations on MutL endonuclease; we discuss the pathological effects of the corresponding mutations in human PMS2.

scholarly journals Discrete mutations introduced in the predicted nucleotide-binding sites of the mdr1 gene abolish its ability to confer multidrug resistance.

1989 ◽  
Vol 9 (12) ◽  
pp. 5289-5297 ◽  
Author(s):  
M Azzaria ◽  
E Schurr ◽  
P Gros
Keyword(s):  
Drug Resistance ◽  
Biological Activity ◽  
Multidrug Resistance ◽  
Amino Acid ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Nucleotide Binding ◽  
Atp Binding ◽  
Nucleotide Binding Sites ◽  
The Core

In cells stably transfected and overexpressing the mouse mdr1 gene, multidrug resistance is associated with an increased ATP-dependent drug efflux. Analysis of the predicted amino acid sequence of the MDR1 protein revealed the presence of two putative nucleotide-binding sites (NBS). To assess the functional importance of these NBS in the overall drug resistance phenotype conferred by mdr1, we introduced amino acid substitutions in the core consensus sequence for nucleotide binding, GXGKST. Mutants bearing the sequence GXAKST or GXGRST at either of the two NBS of mdr1 and a double mutant harboring the sequence GXGRST at both NBS were generated. The integrity of the two NBS was essential for the biological activity of mdr1, since all five mutants were unable to confer drug resistance to hamster drug-sensitive cells in transfection experiments. Conversely, a lysine-to-arginine substitution outside the core consensus sequence had no effect on the activity of mdr1. Failure to reduce intracellular accumulation of [3H]vinblastine paralleled the loss of activity in cell clones expressing mutant MDR1 proteins. However, the ability to bind the photoactivatable ATP analog 8-azido ATP was retained in the five inactive MDR1 mutants. This result implies that an essential step subsequent to ATP binding is impaired in these mutants, possibly ATP hydrolysis or secondary conformational changes induced by ATP-binding or hydrolysis. Our results suggest that the two NBS function in a cooperative fashion, since mutations in a single NBS completely abrogated the biological activity of mdr1.

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