Methyl-directed DNA Mismatch Repair Research Articles

IgA-Dominated Humoral Immune Responses Govern Patients' Outcome in Endometrial Cancer.

This study provides new insights into the crucial role of humoral immunity in human endometrial cancer, providing a rationale for designing novel immunotherapies against this prevalent malignancy. See related commentary by Osorio and Zamarin, p. 766.

Atomic force microscopy captures the initiation of methyl-directed DNA mismatch repair

In Escherichia coli, errors in newly-replicated DNA, such as the incorporation of a nucleotide with a mis-paired base or an accidental insertion or deletion of nucleotides, are corrected by a methyl-directed mismatch repair (MMR) pathway. While the enzymology of MMR has long been established, many fundamental aspects of its mechanisms remain elusive, such as the structures, compositions, and orientations of complexes of MutS, MutL, and MutH as they initiate repair. Using atomic force microscopy, we—for the first time—record the structures and locations of individual complexes of MutS, MutL and MutH bound to DNA molecules during the initial stages of mismatch repair. This technique reveals a number of striking and unexpected structures, such as the growth and disassembly of large multimeric complexes at mismatched sites, complexes of MutS and MutL anchoring latent MutH onto hemi-methylated d(GATC) sites or bound themselves at nicks in the DNA, and complexes directly bridging mismatched and hemi-methylated d(GATC) sites by looping the DNA. The observations from these single-molecule studies provide new opportunities to resolve some of the long-standing controversies in the field and underscore the dynamic heterogeneity and versatility of MutSLH complexes in the repair process.

Role of a short tandem leucine/arginine repeat in strong mutator phenotype acquisition in a clinical isolate ofSalmonellaTyphimurium

In this prospective study, a strong mutator strain of Salmonella Typhimurium was isolated from a collection of 130 human clinical strains of Salmonella. Sequence analysis of the mutS, mutL, and mutH genes, which encode three proteins that are essential for initiation of methyl-directed DNA mismatch repair, revealed insertion of a short tandem repeat (STR) of leucine/alanine in the histidine kinase-like ATPase domain of MutL. The role of this STR in the acquisition of the strong mutator phenotype was confirmed by the construction of an isogenic mutant (6bpinsmutL) from a normomutator strain of Salmonella Heidelberg. This result adds to the sparse body of knowledge about strong mutators and highlights the role of this STR as a hotspot for the acquisition of a strong mutator phenotype in Salmonella.

Single-Molecule Studies of the ssDNA Binding Activity of E. Coli MutL

MutL stimulates the DNA duplex unwinding activity of UvrD in methyl-directed DNA mismatch repair (MMR) via their physical interactions. However, the molecular functions of MutL associated with the DNA binding and UvrD helicase have been partially understood. We present the kinetic characteristics of the single-stranded DNA (ssDNA) binding activity of MutL in the absence or the presence of UvrD helicases using the single-molecule techniques. The lengthening of the ssDNA due to the ssDNA binding of MutL allows us to observe association and dissociation of MutL from the ssDNA in real-time.

Involvement of the β Clamp in Methyl-directed Mismatch Repair in Vitro

We have examined function of the bacterial β replication clamp in the different steps of methyl-directed DNA mismatch repair. The mismatch-, MutS-, and MutL-dependent activation of MutH is unaffected by the presence or orientation of loaded β clamp on either 3′ or 5′ heteroduplexes. Similarly, β is not required for 3′ or 5′ mismatch-provoked excision when scored in the presence of γ complex or in the presence of γ complex and DNA polymerase III core components. However, mismatch repair does not occur in the absence of β, an effect we attribute to a requirement for the clamp in the repair DNA synthesis step of the reaction. We have confirmed previous findings that β clamp interacts specifically with MutS and MutL (López de Saro, F. J., Marinus, M. G., Modrich, P., and O'Donnell, M. (2006) J. Biol. Chem. 281, 14340–14349) and show that the mutator phenotype conferred by amino acid substitution within the MutS N-terminal β-interaction motif is the probable result of instability coupled with reduced activity in multiple steps of the repair reaction. In addition, we have found that the DNA polymerase III α catalytic subunit interacts strongly and specifically with both MutS and MutL. Because interactions of polymerase III holoenzyme components with MutS and MutL appear to be of limited import during the initiation and excision steps of mismatch correction, we suggest that their significance might lie in the control of replication fork events in response to the sensing of DNA lesions by the repair system.

Bacterial genome size reduction by experimental evolution

Bacterial evolution toward endosymbiosis with eukaryotic cells is associated with extensive bacterial genome reduction and loss of metabolic and regulatory capabilities. Here we examined the rate and process of genome reduction in the bacterium Salmonella enterica by a serial passage experimental evolution procedure. The initial rate of DNA loss was estimated to be 0.05 bp per chromosome per generation for a WT bacterium and approximately 50-fold higher for a mutS mutant defective in methyl-directed DNA mismatch repair. The endpoints were identified for seven chromosomal deletions isolated during serial passage and in two separate genetic selections. Deletions ranged in size from 1 to 202 kb, and most of them were not associated with DNA repeats, indicating that they were formed via RecA-independent recombination events. These results suggest that extensive genome reduction can occur on a short evolutionary time scale and that RecA-dependent homologous recombination only plays a limited role in this process of jettisoning superfluous DNA.

Structural basis for MutH activation in E.coli mismatch repair and relationship of MutH to restriction endonucleases

MutS, MutL and MutH are the three essential proteins for initiation of methyl-directed DNA mismatch repair to correct mistakes made during DNA replication in Escherichia coli. MutH cleaves a newly synthesized and unmethylated daughter strand 5' to the sequence d(GATC) in a hemi-methylated duplex. Activation of MutH requires the recognition of a DNA mismatch by MutS and MutL. We have crystallized MutH in two space groups and solved the structures at 1.7 and 2.3 A resolution, respectively. The active site of MutH is located at an interface between two subdomains that pivot relative to one another, as revealed by comparison of the crystal structures, and this presumably regulates the nuclease activity. The relative motion of the two subdomains in MutH correlates with the position of a protruding C-terminal helix. This helix appears to act as a molecular lever through which MutS and MutL may communicate the detection of a DNA mismatch and activate MutH. With sequence homology to Sau3AI and structural similarity to PvuII endonuclease, MutH is clearly related to these enzymes by divergent evolution, and this suggests that type II restriction endonucleases evolved from a common ancestor.

Heterogeneity of DM kinase repeat expansion in different fetal tissues and further expansion during cell proliferation in vitro: evidence for a casual involvement of methyl-directed DNA mismatch repair in triplet repeat stability.

We have analysed the mitotic behaviour of expanded CTG repeats in somatic tissues and cultured somatic cells from myotonic dystrophy (DM) fetuses using indirect and direct methods. Heterogeneity of expansions between fetal tissues was demonstrated in a 16 week old fetus whereas there was no evidence for such a somatic heterogeneity in a 13 week old fetus. Dilution plating of cultured cells from an adult patient and a fetus resulted in isolation of clones showing single expanded restriction fragments when the donor showed a heterogeneous smear of expansions or a single expanded fragment. During proliferation in vitro to 45 doublings, DM cells experienced highly synchronous further repeat expansion which first became evident at approximately 15 cell generations and reached a plateau of maximum expansion at approximately 200 days. When mathematically expressed as a function of culture time the dynamics of expansion of restriction fragments followed a sigmoid curve. This unstable behaviour of CTG repeat expansions in DM was compared to the mitotically stable patterns of full mutation in fragile X fetal tissues and led to the hypothesis that methylation of CpGs within the repeat sequence is a stabilizing factor of largely expanded CGG and GCC repeats allowing for efficient methyl-directed strand-specific DNA mismatch repair.

Methyl directed DNA mismatch repair inVibrio cholerae

Mismatches in DNA occur either due to replication error or during recombination between homologous but non-identical DNA sequences or due to chemical modification of bases. The mismatch in DNA, if not repaired, result in high spontaneous mutation frequency. The repair has to be in the newly synthesized strand of the DNA molecule, otherwise the error will be fixed permanently. Three distinct mechanisms have been proposed for the repair of mismatches in DNA in prokaryotic cells and gene functions involved in these repair processes have been identified. The methyl-directed DNA mismatch repair has been examined inVibrio cholerae, a highly pathogenic gram negative bacterium and the causative agent of the diarrhoeal disease cholera. The DNA adenine methyltransferase encoding gene (dam) of this organism which is involved in strand discrimination during the repair process has been cloned and the complete nucleotide sequence has been determined.Vibrio cholerae dam gene codes for a 21.5 kDa protein and can substitute for theEscherichia coli enzyme. Overproduction ofVibrio cholerae Dam protein is neither hypermutable nor lethal both in Escherichia coli andVibrio cholerae. WhileEscherichia coli dam mutants are sensitive to 2-aminopurine,Vibrio cholerae 2-aminopurine sensitive mutants have been isolated with intact GATC methylation activity. The mutator genesmutS andmutL involved in the recognition of mismatch have been cloned, nucleotide sequence determined and their products characterized. Mutants ofmutS andmutL ofVibrio cholerae have been isolated and show high rate of spontaneous mutation frequency. ThemutU gene ofVibrio cholerae, the product of which is a DNA helicase II, codes for a 70 kDa protein. The deduced amino acid sequence of themutU gene hs all the consensus helicase motifs. The DNA cytosine methyltransferase encoding gene (dam) ofVibrio cholerae has also been cloned. Thedcm gene codes for a 53 kDa protein. This gene product might be involved in very short patch (VSP) repair of DNA mismatches. The vsr gene which is directly involved in VSP repair process codes for a 23 kDa protein. Using these information, the status of DNA mismatch repair inVibrio cholerae will be discussed.

Rates of spontaneous mutation in bacteriophage T4 are independent of host fidelity determinants.

Bacteriophage T4 encodes most of the genes whose products are required for its DNA metabolism, and host (Escherichia coli) genes can only infrequently complement mutationally inactivated T4 genes. We screened the following host mutator mutations for effects on spontaneous mutation rates in T4: mutT (destruction of aberrant dGTPs), polA, polB and polC (DNA polymerases), dnaQ (exonucleolytic proofreading), mutH, mutS, mutL and uvrD (methyl-directed DNA mismatch repair), mutM and mutY (excision repair of oxygen-damaged DNA), mutA (function unknown), and topB and osmZ (affecting DNA topology). None increased T4 spontaneous mutation rates within a resolving power of about twofold (nor did optA, which is not a mutator but overexpresses a host dGTPase). Previous screens in T4 have revealed strong mutator mutations only in the gene encoding the viral DNA polymerase and proofreading 3'-exonuclease, plus weak mutators in several polymerase accessory proteins or determinants of dNTP pool sizes. T4 maintains a spontaneous mutation rate per base pair about 30-fold greater than that of its host. Thus, the joint high fidelity of insertion by T4 DNA polymerase and proofreading by its associated 3'-exonuclease appear to determine the T4 spontaneous mutation rate, whereas the host requires numerous additional systems to achieve high replication fidelity.

The DNA adenine methyltransferase-encoding gene ( dam) of Vibrio cholerae

The DNA adenine methyltransferase (MTase)-encoding gene ( dam) of Vibrio cholerae, an organism belonging to the family Vibrionaceae, has been cloned and the complete nucleotide (nt) sequence determined. V. cholerae dam encodes a 21.5-kDa protein and is directly involved in methyl-directed DNA mismatch repair. It can substitute for the Escherichia coli enzyme and can suppress the phenotypic traits associated with E. coli dam mutants. Overproduction of V. cholerae Dam MTase does not result in hypermutability in either V. cholerae or E. coli cells. Overproduction of V. cholerae Dam in a pUC plasmid, however, fails to suppress the 2-aminopurine (2-AP)-sensitive phenotype of E. coli dam mutants. Homology between the nt and deduced amino acid (aa) sequences of the E. coli and V. cholerae dam genes is only 30–35%.

Isolation and Characterization of the Escherichia coli mutL Gene Product

The Escherichia coli mutL gene product has been purified to near homogeneity from an overproducing clone. The mutL locus encodes a polypeptide of 70,000 daltons as determined by denaturing gel electrophoresis. The native molecular weight of MutL protein as calculated from the sedimentation coefficient of 5.5 S and Stokes radius of 61 A is 139,000 daltons, indicating that MutL exists as a dimer in solution. In addition to its ability to complement methyl-directed DNA mismatch repair in mutL-deficient cell-free extracts, DNase I protection experiments demonstrate that the purified MutL protein interacts with the MutS-heteroduplex DNA complex in the presence of ATP.

Methyl-directed DNA mismatch repair in Escherichia coli

Some of the molecular aspects of methyl-directed mismatch repair in E. coli have been characterized. These include: mismatch recognition by mutS protein in which different mispairs are bound with different affinities; the direct involvement of d(GATC) sites; and strand scission by mutH protein at d(GATC) sequences with strand selection based on methylation of the DNA at those sites. In addition, communication over a distance between a mismatch and d(GATC) sites has been implicated. Analysis of mismatch correlation in a defined system (Lahue et al., unpublished) should provide a direct means to further molecular aspects of this process.

Different base/base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch-repair system of E. coli.

The efficiency of methyl-directed DNA mismatch-repair of E. coli acting in vivo on heteroduplex genomes of phage M13 was found to be strongly dependent on the nature of the base/base mismatch to be corrected. Three efficiency classes were characterized:high (T/G, C/A and G/G); intermediate (A/A); and low (G/A, A/G, T/T, C/C, C/T and T/C). Methyl-directed DNA mismatch repair was lost completely for any type of mismatch in strains carrying either mutL or mutS mutations. Data obtained with a mutH mutant suggest that this locus is involved in methyl-dependent DNA strand discrimination. A functional correlation is suggested between the differential repair efficiencies and the frequencies of the corresponding replication errors.