Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 5 de 5
Filter
Add more filters










Database
Language
Publication year range
1.
Biochem Biophys Res Commun ; 385(2): 225-9, 2009 Jul 24.
Article in English | MEDLINE | ID: mdl-19450548

ABSTRACT

The effect of wild-type and mutant MutL on the steady-state ATPase activity of MutS from Escherichia coli has been investigated in the absence and presence of 22, 50, and 75 base pair hetero- and homoduplex DNAs with open and blocked ends. The steady-state ATPase activity of MutS has been measured at 37 degrees C using a spectrophotometric method. The presence of MutL did not affect appreciably on the ATPase activity of MutS in the absence of DNA or in the presence of blocked end homoduplex DNAs. However, the addition of MutL affected oppositely on the ATPase activity of MutS in the presence of G-T mismatched DNAs depending on their end status. We have also found that only the ATPase active forms of MutL increased the ATPase activity of MutS in the presence of G-T mismatched DNAs with blocked ends. The results suggest that MutL ATPase activity is required to catalyze dissociation of the MutS sliding clamps.


Subject(s)
Adenosine Triphosphatases/metabolism , DNA Mismatch Repair , DNA/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , MutS DNA Mismatch-Binding Protein/metabolism , Adenosine Triphosphatases/genetics , Catalysis , DNA/genetics , DNA Replication , Escherichia coli/genetics , Escherichia coli Proteins/genetics , MutL Proteins
2.
BMB Rep ; 42(1): 53-8, 2009 Jan 31.
Article in English | MEDLINE | ID: mdl-19192394

ABSTRACT

The methyl-directed mismatch repair (MMR) mechanism has been extensively studied in vitro and in vivo, but one of the difficulties in determining the biological relationships between the MMR-related proteins is the tendency of MutL to self-aggregate. The properties of a stable MutL homologue were investigated using a thermostable MutL (TmL) from Thermotoga maritima MSB8 and whose size exclusion chromatographic and crosslinking analyses were compatible with a dimeric form of TmL. TmL underwent conformational changes in the presence of nucleotides and single-stranded DNA (ssDNA) with ATP binding not requiring ssDNA binding activity of TmL, while ADPnP-stimulated TmL showed a high ssDNA binding affinity. Finally, TmL interacted with the T. maritima MutS (TmS), increasing the affinity of TmS to mismatched DNA base pairs and suggesting that the role of TmL in the formation of a mismatched DNA-TmS complex may be a pivotal observation for the study of the initial MMR system. [BMB reports 2009; 42(1): 53-58].


Subject(s)
Bacterial Proteins/metabolism , Temperature , Thermotoga maritima/metabolism , Adenosine Triphosphate/metabolism , Base Pair Mismatch/drug effects , Chromatography, Gel , Cross-Linking Reagents/pharmacology , DNA, Bacterial/metabolism , Electrophoretic Mobility Shift Assay , Hydrolysis/drug effects , Kinetics , Mutant Proteins/metabolism , Nucleotides/metabolism , Protein Binding/drug effects , Protein Conformation , Protein Multimerization/drug effects , Protein Stability/drug effects , Thermotoga maritima/drug effects
3.
J Biochem ; 145(2): 199-206, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19029144

ABSTRACT

MutL is required to assist the mismatch repair protein MutS during initiation of the methyl-directed mismatch repair (MMR) response in various organisms ranging from prokaryotes to eukaryotes. Despite this necessity, the inherent propensity of MutL to aggregate has led to significant difficulties in determining its biological relationship with other MMR-related proteins. Here, we perform analysis on the thermostable MutL protein found in Thermotoga maritima MSB8 (TmL). Size exclusion chromatographic analysis indicates the lack of aggregated forms with the exception of a dimeric TmL. Small-angle X-ray scattering (SAXS) analysis reveals that the solution structures of the full-length TmL and its corresponding complexes with nucleotides and ssDNA undergo conformational changes. The elucidated TmL SAXS model is superimposed to the crystal structure of the C-terminal domain of Escherichia coli MutL. In addition, the N-terminal SAXS model of TmL exists as monomeric form, indicating that TmL has a structurally flexible N-terminal domain. TmL SAXS analysis can suggest a considerable possibility on a new 3D view of the previously unresolved full-length MutL molecule.


Subject(s)
Adenosine Triphosphatases/chemistry , Bacterial Proteins/chemistry , Nucleotides/metabolism , Thermotoga maritima/metabolism , Amino Acid Sequence , Binding Sites , Chromatography, Gel , DNA Mismatch Repair , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/metabolism , Dimerization , Escherichia coli Proteins/chemistry , Models, Molecular , Molecular Sequence Data , MutL Proteins , Protein Conformation , Sequence Alignment
4.
Biochem Biophys Res Commun ; 364(2): 264-9, 2007 Dec 14.
Article in English | MEDLINE | ID: mdl-17950245

ABSTRACT

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.


Subject(s)
Adenosine Triphosphatases/chemistry , DNA, Bacterial/chemistry , Escherichia coli Proteins/chemistry , MutS DNA Mismatch-Binding Protein/chemistry , Nucleic Acid Heteroduplexes/chemistry , Enzyme Activation
5.
Biosens Bioelectron ; 22(7): 1376-81, 2007 Feb 15.
Article in English | MEDLINE | ID: mdl-16876990

ABSTRACT

A fluorescent method was developed for the detection of unpaired and mismatched DNAs using a MutS-fluorophore conjugate. The fluorophore, 2-(4'-(iodoacetoamido)anilino) naphthalene-6-sulfonic acid (IAANS), was site-specifically attached to the 469 position of Thermus aquaticus (Taq.) MutS mutant (C42A/T469C). The fluorophore labeled residue located at the dimer interface of the protein undergoes a drastic conformational change upon binding with mismatched DNA. The close proximity of the two identical fluorescent molecules presumably causes the self-quenching of the fluorophore, since fluorescence emission of the biosensor decreases with increasing concentrations of mismatched DNA. The order of binding affinity for each unpaired and mismatched DNA obtained by this method was DeltaT (Kd=52 nM)>GT (62 nM)>DeltaC (130 nM)>CT (160 nM)>DeltaG (170 nM)>DeltaA (250 nM)>CC (720 nM)>AT (950 nM). This order is comparable to the previous results of the gel mobility shift assay. Thus, this method can be a simple, useful tool for elucidating the mechanism of DNA mismatch repair as well as a novel probe for detecting of genetic mutation.


Subject(s)
DNA/analysis , DNA/chemistry , Fluorescent Dyes , MutS DNA Mismatch-Binding Protein , Biosensing Techniques , MutS DNA Mismatch-Binding Protein/genetics , Naphthalenesulfonates , Point Mutation , Thermus
SELECTION OF CITATIONS
SEARCH DETAIL
...