The DNA Binding Activity of MutL Is Required for Methyl-directed Mismatch Repair in Escherichia coli

Abstract

The DNA binding properties of the mismatch repair protein MutL and their importance in the repair process have been controversial for nearly two decades. We have addressed this issue using a point mutant of MutL (MutL-R266E). The biochemical and genetic data suggest that DNA binding by MutL is required for dam methylation-directed mismatch repair. 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 with MutL, and consistent with this observation, its DNA-dependent biochemical activities, like DNA-stimulated ATP hydrolysis and helicase II stimulation, are severely compromised. In addition, there is a modest effect on stimulation of MutH-catalyzed nicking. Finally, genetic assays show that MutL-R266E has a strong mutator phenotype, demonstrating that the mutant is unable to function in dam methylation-directed mismatch repair in vivo. Methyl-directed mismatch repair (MMR)2 is the primary pathway for correcting replication errors and unwanted recombination events in the Escherichia coli bacterial cell (1-3). Therefore, a functional MMR pathway is essential for ensuring the integrity of the chromosome as well as for 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 Exo I and Exo X (3Ј to 5Ј polarity), Exo VII and RecJ (5Ј to 3Ј polarity) (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 Refs. 2 and 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 an ␣-shaped loop in the DNA (14) that is dependent on ATP and thought to be the mode for communicating the signal from the mismatch to the nearest hemi-methylated d(GATC) site, where MutH binds to provide the strand discrimination essential in methyl-directed mismatch repair. Once the MutS-MutL complex locates a MutHbound 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 hemi-methylated 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 3Ј to 5Ј direction (18), unwinding the DNA duplex until the mismatch has been removed (19). The signal indicating that sufficient DNA has been...

DNA mismatch repair: Molecular mechanism, cancer, and ageing

“…A crystal structure of a C-terminal dimerization domain of E. coli MutL reveals a V-shaped dimer that plays critical roles in the activation of UvrD by MutL and in DNA binding by MutL (Guarne et al, 2004). The DNA binding activity of MutL has been shown to be essential for methyl-directed MMR in E. coli (Robertson et al, 2006a). The N-terminal ATPase domain and the C-terminal dimerization domain are connected to each other via a flexible peptide linker with the elbow of the dimerization domain at one end and the lid of the ATPase domain at the other.…”

Functional properties of the thermostable mutL from Thermotoga maritima

“…E. coli MutL can bind to a 93 bp partial duplex DNA with an apparent KD of 25 and 2.8 nM in the absence and presence of ADPnP, respectively (10). The investigation of the DNA binding affinity of TmL by a double-filter method performed using 30-mer ssDNA and dsDNA showed the μM range of Kd for TmL binding to a 30 bp dsDNA in the absence or presence of ADPnP.…”

“…Previous studies have indicated that MutL binds to ssDNA with nucleotide correlation (9,10). E. coli MutL can bind to a 93 bp partial duplex DNA with an apparent KD of 25 and 2.8 nM in the absence and presence of ADPnP, respectively (10).…”

“…As a mediator in the MMR pathway, MutL possesses a relatively weak ATP hydrolytic activity, compared to other ATPase proteins (7), as well as a nonspecific DNA binding activity, especially to single-stranded DNA (ssDNA) (8)(9)(10). X-ray crystallographic data suggests that MutL ATP binding and hydrolysis causes major conformational changes which affect interactions with other MMR-related proteins (11,12).…”

“…X-ray crystallographic data suggests that MutL ATP binding and hydrolysis causes major conformational changes which affect interactions with other MMR-related proteins (11,12). Since the inclusion of ssDNA can stimulate MutL-catalyzed ATP hydrolysis, many studies have examined the correlation between the DNA binding properties of MutL and its ATPase activity (8,10).…”