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Table 1. Mismatch repair proteins
E. coli Yeast Human Plants Dimerization partner
MutL PMS1 PMS2 PMS2 Heterodimerizes with MLH1
MLH1 MLH1 MLH1 Heterodimerizes with PMS1, MLH2, and MLH3 MLH2 PMS1 N/A Heterodimerizes with MLH1
MLH3 MLH3 MLH3 Heterodimerizes with MLH1
MutS MSH1 N/A MSH1 Mitochondrial MMR
MSH2 MSH2 MSH2 Heterodimerizes with MSH3 and MSH6 MSH3 MSH3 MSH3 Heterodimerizes with MSH2
MSH4 MSH4 MSH4 Heterodimerizes with MSH5 MSH5 MSH5 MSH5 Heterodimerizes with MSH4 MSH6 MSH6 MSH6 Heterodimerizes with MSH2
MSH7 Unknown
MutH N/A MLH1-PMS2 N/A
UvrD N/A N/A N/A
ExoI N/A ExoI N/A
ExoX N/A ExoI N/A
RecJ 5' to 3' Exonuclease of Pol δ
ExoVII 5' to 3' Exonuclease of Pol δ
DNA Pol III Pol δ
SSB RPA
DNA Ligase I DNA Ligase
(Adapted from Kunkel, T.A. and Erie, D. A. Annu. Rev. Biochem. 2005;74:681-710)
Figure 2. Model for Human DNA Mismatch Repair (Taken from Modrich, P. J. Biol. Chem. 2006;281:30305-30309)
Chapter 2
The DNA Binding Activity of MutL is Essential for Methyl-directed Mismatch Repair in Escherichia coli
The DNA binding properties of the DNA mismatch repair protein MutL and their importance in the repair process have been controversial for nearly two decades. We have shown, through the use of a single point mutant of MutL (MutL-R266E), that DNA binding by MutL is required for dam-directed mismatch repair in vivo. We demonstrate that purified MutL-R266E retains wild-type biochemical properties that do not depend on DNA binding, such as basal ATP hydrolysis in the absence of DNA and the ability to interact with other mismatch repair proteins. However, purified MutL-R266E binds DNA poorly in vitro as compared to MutL and, consistent with this observation, its DNA-dependent biochemical activities, like helicase II stimulation and DNA-stimulated ATP hydrolysis, are severely affected. Finally, genetic assays show that MutL-R266E has a strong mutator phenotype demonstrating that the mutant is unable to function in dam-directed mismatch repair in vivo. Based upon the evidence collected we have determined that DNA binding is essential for mismatch repair function.
Introduction
Methyl-directed mismatch repair (MMR) is the primary pathway for correcting replication errors and unwanted recombination events in the E. coli bacterial cell (1-3). Therefore, a functional MMR pathway is essential for ensuring the integrity of the chromosome, as well as maintaining an acceptable cellular mutation rate. The primary components of the bacterial MMR system are the mutator proteins (MutL, MutS, MutH, and UvrD), several exonucleases, including ExoI, ExoVII, and RecJ (4-6), DNA polymerase III, single-stranded DNA binding protein and DNA ligase. These proteins act in concert to correct base-base mismatches after passage of the replication fork (7).
A current model of MMR (for reviews see (2,8-10) posits that the MutS protein recognizes and binds the mismatched base pair, followed by the binding of MutL to form a ternary complex (11). In E. coli both of these proteins function as homodimers (12,13). This complex forms a loop in the DNA (14) in search of the nearest hemi-methlyated d(GATC) site where MutH binds to provide the strand discrimination essential in methyl-directed mismatch repair. Once the MutS-MutL complex locates a MutH-bound hemi-methylated d(GATC) site, MutH is stimulated, presumably by MutL (15,16), to nick the unmethylated DNA strand resulting in discrimination of the nascent and parental DNA strands (4,17). The use of the nearest hemimethylated d(GATC) as the site of MutH-directed incision provides MMR with a bidirectional capability since this site could be located on either side of the mismatch. Helicase II (UvrD gene product) is then loaded on the appropriate strand at the nick and translocates in a 3’ to 5’ direction (18) unwinding the DNA duplex until the mismatch has been removed (19). The signal indicating sufficient DNA has been unwound to complete the repair process is not
known. One of the several nucleases, with an appropriate polarity, degrades the nascent DNA strand and single-stranded DNA binding protein binds and stabilizes the single-stranded DNA (ssDNA) template until DNA polymerase III is recruited to fill the gap. The resulting nick is sealed by DNA ligase, completing the repair process and restoring the integrity of the DNA (13).
The role of MutL protein in MMR remains to be completely defined on a mechanistic level. The protein was originally purified, using a biochemical complementation assay, as an essential component for partially reconstituted mismatch repair in cell extracts lacking MutL (12). Subsequent experiments demonstrated its interaction with MutS at a mismatch (13,20) and the solved crystal structure of the amino-terminal domain of MutL demonstrated an ATP binding/hydrolysis fold common to the GHKL group of ATP hydrolyzing enzymes (21). Purified MutL catalyzes a slow ATP hydrolysis reaction that is stimulated by the presence of ssDNA and is essential for MMR (20-22). In addition, MutL has been shown to interact with and stimulate the hemi-methylated d(GATC)-directed nicking reaction catalyzed by MutH (15,16) as well stimulating the unwinding catalyzed by UvrD (19). Thus, MutL has been characterized as the master regulator of MMR in view of its interaction with many of the proteins required for MMR.
The stimulation of MutL-catalyzed ATP hydrolysis by the addition of DNA has prompted an investigation of the DNA binding properties of MutL. Several groups have demonstrated that MutL binds to both ssDNA and double-stranded DNA (dsDNA) (20,23), while others report that MutL does not bind DNA (24). Recently the DNA binding activity of MutL has been suggested to be an artifact of in vitro assays designed to measure DNA binding (25). Indeed,
a current model for MMR postulates that DNA binding by MutL is not required for the MMR processes (26).
Using a combination of biochemical and genetic assays we present evidence to suggest that DNA binding by MutL is an absolute requirement for MMR. These studies take advantage of a mutL point mutant (MutL-R266E) in which the arginine at position 266 has been altered to a glutamic acid. Expression of this protein in a cell strain lacking functional MutL results in a mutator phenotype similar to that observed with a complete deletion of the mutL gene. The purified mutant protein, MutL-R266E, exhibits a weak ssDNA binding affinity, a weak ssDNA-stimulated ATPase activity and poor stimulation of UvrD-catalyzed DNA unwinding. Taken together, these data are consistent with a primary DNA binding defect since the purified protein is able to interact with the other MMR proteins as effectively as wild-type MutL protein. We conclude that the DNA binding activity of MutL is essential for MMR.
Materials and Methods
Bacterial strains and plasmids – E. coli BL21(DE3) (F- ompT [lon] hsdSBrB-mB-gal λDE3)
was from Novagen. A derivative of the strain, BL21(DE3)uvrD::Tn5 mutL::Tn10 has been described previously (27). The MutL gene was cloned into the pET15b expression vector (Novagen) by removing the mutL gene from pET3C-MutL (28) with BamHI and inserting the gene in pET15b cut with the same restriction enzyme. This plasmid is referred to as pET15b-MutL. The pET15b-MutL-R266E construct was a gift from Peggy Hsieh (National Institutes of Health). Constructs were sequenced to verify the coding sequence of the gene and to ensure the absence of any unintended mutations.
Protein Purification – MutL and MutL-R266E were expressed in E. coli BL21(DE3) uvrD::Tn5 mutL::Tn10 cells containing the appropriate MutL expression vector. Two liters of cells were grown at 37oC to an A600 of 0.8 and protein expression was induced by the
adding IPTG to 0.5 mM. After an additional four hour incubation at 37oC the cells were harvested by centrifugation, washed with cold H2O, suspended in binding buffer (50 mM
NaPO4 (pH 7.0), 500 mM NaCl, 5 mM imidazole, 10 % (v/v) glycerol) and frozen at –75oC
until future use. Cells were thawed on ice and lysed by the addition of lysozyme to 200
μg/ml followed by incubation at 0oC for 60 min. The lysate was sonciated briefly to reduce the viscosity, applied to a TALON metal affinity resin (BD Biosciences - 1 ml of resin/L cells) and extensively washed with binding buffer. To decrease the NaCl concentration before elution, the column was washed with several column volumes of binding buffer containing 225 mM NaCl instead of 500 mM NaCl. The column was eluted using 200 mM imidazole in the low NaCl binding buffer. This step results in a substantial purification of