Regulation of UvrD Helicase Activity by MutL

Escherichia coli UvrD is a superfamily 1 helicase/translocase involved in multiple DNA metabolic processes including methyl-directed mismatch DNA repair. Although a UvrD monomer can translocate along single-stranded DNA, a UvrD dimer is needed for processive helicase activity in vitro. E. coli MutL, a regulatory protein involved in methyl-directed mismatch repair, stimulates UvrD helicase activity; however, the mechanism is not well understood. Using single-molecule fluorescence and ensemble approaches, we find that a single MutL dimer can activate latent UvrD monomer helicase activity. However, we also find that MutL stimulates UvrD dimer helicase activity. We further find that MutL enhances the DNA-unwinding processivity of UvrD. Hence, MutL acts as a processivity factor by binding to and presumably moving along with UvrD to facilitate DNA unwinding.     

Evidence for a physical interaction between the Escherichia coli methyl-directed mismatch repair proteins MutL and UvrD.

UvrD (DNA helicase II) is an essential component of two major DNA repair pathways in Escherichia coli: methyl-directed mismatch repair and UvrABC-mediated nucleotide excision repair. In addition, it has an undefined role in the RecF recombination pathway and possibly in replication. In an effort to better understand the role of UvrD in these various aspects of DNA metabolism, a yeast two-hybrid screen was used to search for interacting protein partners. Screening of an E.coli genomic library revealed a potential interaction between UvrD and MutL, a component of the methyl-directed mismatch repair pathway. The interaction was confirmed by affinity chromatography using purified proteins. Deletion analysis demonstrated that the C-terminal 218 amino acids (residues 398-615) of MutL were sufficient to produce the two-hybrid interaction with UvrD. On the other hand, both the N- and C-termini of UvrD were required for interaction with MutL. The implications of this interaction for the mismatch repair mechanism are discussed.     

Escherichia coli MutL loads DNA helicase II onto DNA

Previous studies have shown that MutL physically interacts with UvrD (DNA helicase II) (Hall, M. C., Jordan, J. R., and Matson, S. W. (1998) EMBO J. 17, 1535-1541) and dramatically stimulates the unwinding reaction catalyzed by UvrD in the presence and absence of the other protein components of the methyl-directed mismatch repair pathway (Yamaguchi, M., Dao, V., and Modrich, P. (1998) J. Biol. Chem. 273, 9197-9201). The mechanism of this stimulation was investigated using DNA binding assays, single-turnover helicase assays, and unwinding assays involving long duplex DNA substrates. The results indicate that MutL binds DNA and loads UvrD onto the DNA substrate. The interaction between MutL and DNA and that between MutL and UvrD are both important for stimulation of UvrD-catalyzed unwinding. MutL does not clamp UvrD onto the substrate; and therefore, the processivity of unwinding is not increased in the presence of MutL. The implications of these results are discussed, and models are presented for the mechanism of MutL stimulation as well as for the role of MutL as a master coordinator in the methyl-directed mismatch repair pathway.     

MutL-catalyzed ATP hydrolysis is required at a post-UvrD loading step in methyl-directed mismatch repair

Methyl-directed mismatch repair is a coordinated process that ensures replication fidelity and genome integrity by resolving base pair mismatches and insertion/deletion loops. This post-replicative event involves the activities of several proteins, many of which appear to be regulated by MutL. MutL interacts with and modulates the activities of MutS, MutH, UvrD, and perhaps other proteins. The purified protein catalyzes a slow ATP hydrolysis reaction that is essential for its role in mismatch repair. However, the role of the ATP hydrolysis reaction is not understood. We have begun to address this issue using two point mutants: MutL-E29A, which binds nucleotide but does not catalyze ATP hydrolysis, and MutL-D58A, which does not bind nucleotide. As expected, both mutants failed to complement the loss of MutL in genetic assays. Purified MutL-E29A protein interacted with MutS and stimulated the MutH-catalyzed nicking reaction in a mismatch-dependent manner. Importantly, MutL-E29A stimulated the loading of UvrD on model substrates. In fact, stimulation of UvrD-catalyzed unwinding was more robust with MutL-E29A than the wild-type protein. MutL-D58A, on the other hand, did not interact with MutS, stimulate MutH-catalyzed nicking, or stimulate the loading of UvrD. We conclude that ATP-bound MutL is required for the incision steps associated with mismatch repair and that ATP hydrolysis by MutL is required for a step in the mismatch repair pathway subsequent to the loading of UvrD and may serve to regulate helicase loading.     

Physical and functional interactions between Escherichia coli MutL and the Vsr repair endonuclease

DNA mismatch repair (MMR) and very-short patch (VSP) repair are two pathways involved in the repair of T:G mismatches. To learn about competition and cooperation between these two repair pathways, we analyzed the physical and functional interaction between MutL and Vsr using biophysical and biochemical methods. Analytical ultracentrifugation reveals a nucleotide-dependent interaction between Vsr and the N-terminal domain of MutL. Using chemical crosslinking, we mapped the interaction site of MutL for Vsr to a region between the N-terminal domains similar to that described before for the interaction between MutL and the strand discrimination endonuclease MutH of the MMR system. Competition between MutH and Vsr for binding to MutL resulted in inhibition of the mismatch-provoked MutS- and MutL-dependent activation of MutH, which explains the mutagenic effect of Vsr overexpression. Cooperation between MMR and VSP repair was demonstrated by the stimulation of the Vsr endonuclease in a MutS-, MutL- and ATP-hydrolysis-dependent manner, in agreement with the enhancement of VSP repair by MutS and MutL in vivo. These data suggest a mobile MutS–MutL complex in MMR signalling, that leaves the DNA mismatch prior to, or at the time of, activation of downstream effector molecules such as Vsr or MutH.     

Structure of the MutL C-terminal domain: a model of intact MutL and its roles in mismatch repair

MutL assists the mismatch recognition protein MutS to initiate and coordinate mismatch repair in species ranging from bacteria to humans. The MutL N-terminal ATPase domain is highly conserved, but the C-terminal region shares little sequence similarity among MutL homologs. We report here the crystal structure of the Escherichia coli MutL C-terminal dimerization domain and the likelihood of its conservation among MutL homologs. A 100-residue proline-rich linker between the ATPase and dimerization domains, which generates a large central cavity in MutL dimers, tolerates sequence substitutions and deletions of one-third of its length with no functional consequences in vivo or in vitro. Along the surface of the central cavity, residues essential for DNA binding are located in both the N- and C-terminal domains. Each domain of MutL interacts with UvrD helicase and is required for activating the helicase activity. The DNA-binding capacity of MutL is correlated with the level of UvrD activation. A model of how MutL utilizes its ATPase and DNA-binding activities to mediate mismatch-dependent activation of MutH endonuclease and UvrD helicase is proposed.     

Single-molecule analysis reveals the kinetics and physiological relevance of MutL-ssDNA binding

DNA binding by MutL homologs (MLH/PMS) during mismatch repair (MMR) has been considered based on biochemical and genetic studies. Bulk studies with MutL and its yeast homologs Mlh1-Pms1 have suggested an integral role for a single-stranded DNA (ssDNA) binding activity during MMR. We have developed single-molecule Förster resonance energy transfer (smFRET) and a single-molecule DNA flow-extension assays to examine MutL interaction with ssDNA in real time. The smFRET assay allowed us to observe MutL-ssDNA association and dissociation. We determined that MutL-ssDNA binding required ATP and was the greatest at ionic strength below 25 mM (K_D = 29 nM) while it dramatically decreases above 100 mM (K_D>2 µM). Single-molecule DNA flow-extension analysis suggests that multiple MutL proteins may bind ssDNA at low ionic strength but this activity does not enhance stability at elevated ionic strengths. These studies are consistent with the conclusion that a stable MutL-ssDNA interaction is unlikely to occur at physiological salt eliminating a number of MMR models. However, the activity may infer some related dynamic DNA transaction process during MMR.     

DNA mismatch repair in trinucleotide repeat instability

Trinucleotide repeat expansions cause over 30 severe neuromuscular and neurodegenerative disorders, including Huntington's disease, myotonic dystrophy type 1, and fragile X syndrome. Although previous studies have substantially advanced the understanding of the disease biology, many key features remain unknown. DNA mismatch repair(MMR) plays a critical role in genome maintenance by removing DNA mismatches generated during DNA replication. However, MMR components,particularly mismatch recognition protein MutSβ and its interacting factors MutLα and MutLγ, have been implicated in trinucleotide repeat instability. In this review, we will discuss the roles of these key MMR proteins in promoting trinucleotide repeat instability.     

The PMS2 subunit of human MutLalpha contains a metal ion binding domain of the iron-dependent repressor protein family

DNA mismatch repair (MMR) is responsible for correcting replication errors. MutLα, one of the main players in MMR, has been recently shown to harbor an endonuclease/metal-binding activity, which is important for its function in vivo. This endonuclease activity has been confined to the C-terminal domain of the hPMS2 subunit of the MutLα heterodimer. In this work, we identify a striking sequence-structure similarity of hPMS2 to the metal-binding/dimerization domain of the iron-dependent repressor protein family and present a structural model of the metal-binding domain of MutLα. According to our model, this domain of MutLα comprises at least three highly conserved sequence motifs, which are also present in most MutL homologs from bacteria that do not rely on the endonuclease activity of MutH for strand discrimination. Furthermore, based on our structural model, we predict that MutLα is a zinc ion binding protein and confirm this prediction by way of biochemical analysis of zinc ion binding using the full-length and C-terminal domain of MutLα. Finally, we demonstrate that the conserved residues of the metal ion binding domain are crucial for MMR activity of MutLα in vitro.     

Yeast mutator phenotype enforced by Arabidopsis PMS1 expression

The DNA mismatch repair (MMR) system is a major DNA repair pathway whose function is critical for the correction of DNA biosynthetic errors. MMR is initiated by the binding of MutS proteins to mismatches and unpaired nucleotides followed by the recruitment of MutL proteins. The major MutL activity in eukaryotes is performed by MutLα, the heterocomplex of MLH1-PMS1 in yeast and plants and MLH1-PMS2 in humans. We here report the effect the expression of Arabidopsis PMS1 protein exerts on Saccharomyces cerevisiae genomic stability. A strain carrying specific microsatellite instability reporter systems was chosen for the study. The plant protein failed to complement the hypermutator phenotype of a pms1 deficient strain but increased approximately 14-fold and 2,000-fold the mutation rates of his7-2 and lys2::InsE-A ₁₄ loci of MMR proficient strains when compared to wild-type strains, respectively. Overexpressing AtMLH1 in the AtPMS1-overproducing strain generated an increase in mutation rate comparable to that of AtPMS1 expression alone. Deletion of the C-terminal residues implicated in protein–protein interaction and including the putative endonuclease sequence of AtPMS1 completely eliminated the mutator phenotype. Taken together, these results indicate that the plant proteins affect yeast genomic stability, very possibly altering protein–protein interactions that are necessary to complete repair.     

Mismatch repair ensures fidelity of replication and recombination in the radioresistant organism<Emphasis Type="Italic"> Deinococcus radiodurans</Emphasis>

We have characterized the mismatch repair system (MMR) of the highly radiation-resistant type strain of Deinococcus radiodurans, ATCC 13939. We show that the MMR system is functional in this organism, where it participates in ensuring the fidelity of DNA replication and recombination. The system relies on the activity of two key proteins, MutS1 and MutL, which constitute a conserved core involved in mismatch recognition. Inactivation of MutS1 or MutL resulted in a seven-fold increase in the frequency of spontaneous Rif^R mutagenesis and a ten-fold increase in the efficiency of integration of a donor point-mutation marker during bacterial transformation. Inactivation of the mismatch repair-associated UvrD helicase increased the level of spontaneous mutagenesis, but had no effect on marker integration—suggesting that binding of MutS1 and MutL proteins to a mismatched heteroduplex suffices to inhibit recombination between non identical (homeologous) DNAs. In contrast, inactivation of MutS2, encoded by the second mutS -related gene present in D. radiodurans, had no effect on mutagenesis or recombination. Cells devoid of MutS1 or MutL proteins were as resistant to -rays, mitomycin C and UV-irradiation as wild-type bacteria, suggesting that the mismatch repair system is not essential for the reconstitution of a functional genome after DNA damage.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by G. Baldacci     

Stoichiometry of MutS and MutL at unrepaired mismatches in vivo suggests a mechanism of repair

Mismatch repair (MMR) is an evolutionarily conserved DNA repair system, which corrects mismatched bases arising during DNA replication. MutS recognizes and binds base pair mismatches, while the MutL protein interacts with MutS-mismatch complex and triggers MutH endonuclease activity at a distal-strand discrimination site on the DNA. The mechanism of communication between these two distal sites on the DNA is not known. We used functional fluorescent MMR proteins, MutS and MutL, in order to investigate the formation of the fluorescent MMR protein complexes on mismatches in real-time in growing Escherichia coli cells. We found that MutS and MutL proteins co-localize on unrepaired mismatches to form fluorescent foci. MutL foci were, on average, 2.7 times more intense than the MutS foci co-localized on individual mismatches. A steric block on the DNA provided by the MutHE56A mutant protein, which binds to but does not cut the DNA at the strand discrimination site, decreased MutL foci fluorescence 3-fold. This indicates that MutL accumulates from the mismatch site toward strand discrimination site along the DNA. Our results corroborate the hypothesis postulating that MutL accumulation assures the coordination of the MMR activities between the mismatch and the strand discrimination site.     

Spatial coupling between DNA replication and mismatch repair in Caulobacter crescentus

The DNA mismatch repair (MMR) process detects and corrects replication errors in organisms ranging from bacteria to humans. In most bacteria, it is initiated by MutS detecting mismatches and MutL nicking the mismatch-containing DNA strand. Here, we show that MMR reduces the appearance of rifampicin resistances more than a 100-fold in the Caulobacter crescentus Alphaproteobacterium. Using fluorescently-tagged and functional MutS and MutL proteins, live cell microscopy experiments showed that MutS is usually associated with the replisome during the whole S-phase of the C. crescentus cell cycle, while MutL molecules may display a more dynamic association with the replisome. Thus, MMR components appear to use a 1D-scanning mode to search for rare mismatches, although the spatial association between MutS and the replisome is dispensible under standard growth conditions. Conversely, the spatial association of MutL with the replisome appears as critical for MMR in C. crescentus, suggesting a model where the β-sliding clamp licences the endonuclease activity of MutL right behind the replication fork where mismatches are generated. The spatial association between MMR and replisome components may also play a role in speeding up MMR and/or in recognizing which strand needs to be repaired in a variety of Alphaproteobacteria.     

The coordinated functions of the E. coli MutS and MutL proteins in mismatch repair

The Escherichia coli MutS and MutL proteins have been conserved throughout evolution, although their combined functions in mismatch repair (MMR) are poorly understood. We have used biochemical and genetic studies to ascertain a physiologically relevant mechanism for MMR. The MutS protein functions as a regional lesion sensor. ADP-bound MutS specifically recognizes a mismatch. Repetitive rounds of mismatch-provoked ADP-->ATP exchange results in the loading of multiple MutS hydrolysis-independent sliding clamps onto the adjoining duplex DNA. MutL can only associate with ATP-bound MutS sliding clamps. Interaction of the MutS-MutL sliding clamp complex with MutH triggers ATP binding by MutL that enhances the endonuclease activity of MutH. Additionally, MutL promotes ATP binding-independent turnover of idle MutS sliding clamps. These results support a model of MMR that relies on two dynamic and redundant ATP-regulated molecular switches.     

The C-terminal domain of the MutL homolog from Neisseria gonorrhoeae forms an inverted homodimer

The mismatch repair (MMR) pathway serves to maintain the integrity of the genome by removing mispaired bases from the newly synthesized strand. In E. coli, MutS, MutL and MutH coordinate to discriminate the daughter strand through a mechanism involving lack of methylation on the new strand. This facilitates the creation of a nick by MutH in the daughter strand to initiate mismatch repair. Many bacteria and eukaryotes, including humans, do not possess a homolog of MutH. Although the exact strategy for strand discrimination in these organisms is yet to be ascertained, the required nicking endonuclease activity is resident in the C-terminal domain of MutL. This activity is dependent on the integrity of a conserved metal binding motif. Unlike their eukaryotic counterparts, MutL in bacteria like Neisseria exist in the form of a homodimer. Even though this homodimer would possess two active sites, it still acts a nicking endonuclease. Here, we present the crystal structure of the C-terminal domain (CTD) of the MutL homolog of Neisseria gonorrhoeae (NgoL) determined to a resolution of 2.4 Å. The structure shows that the metal binding motif exists in a helical configuration and that four of the six conserved motifs in the MutL family, including the metal binding site, localize together to form a composite active site. NgoL-CTD exists in the form of an elongated inverted homodimer stabilized by a hydrophobic interface rich in leucines. The inverted arrangement places the two composite active sites in each subunit on opposite lateral sides of the homodimer. Such an arrangement raises the possibility that one of the active sites is occluded due to interaction of NgoL with other protein factors involved in MMR. The presentation of only one active site to substrate DNA will ensure that nicking of only one strand occurs to prevent inadvertent and deleterious double stranded cleavage.     

重组 E.coli 解旋酶Ⅱ（UvrD）的DNA结合及解螺旋功能分析

E.coli 解旋酶Ⅱ（UvrD）是一种在甲基定向错配修复（methyl-directed mismatch repair, MMR）和核苷酸切除修复（nucleotide excision repair,NER）中起重要作用的3′→5′解旋酶.本研究对大肠杆菌的UvrD进行了重组表达和纯化,并检测其ATP酶比活性（87　U/mg）. 利用表面等离子共振（surface plasmon resonance, SPR）方法实时检测了UvrD与同源双链DNA分子（homoduplex DNA）和异源双链DNA分子（heteroduplex DNA）结合的动态过程以及镁离子对此过程的影响.结果显示,UvrD与DNA的平衡解离常数在10 ^－7mol/L 水平. DNA分子中错配碱基的存在,在一定程度上影响了二者的结合,而镁离子不是两者结合的必要因素.本研究还利用原子力显微镜（atomic force microscopy,AFM）方法在单分子水平上观察到UvrD将双链DNA解链形成单链DNA的中间体.此研究得到的UvrD与DNA结合的动力学信息数据以及解螺旋中间体的单分子可视化,为进一步深入研究UvrD在修复过程中的作用机制奠定了基础.     

A novel interaction between human DNA polymerase η and MutLα

Human DNA polymerase η (Polη) is the gene product underlying xeroderma pigmentosum variant, and plays principal roles in translesion DNA synthesis. Here, we identified human MLH1, an essential component of mismatch repair (MMR), as a Polη-interacting protein. The middle area residues, which include the little finger domain, of Polη are important for the interaction with MLH1. Polη also interacts with the MLH1/PMS2 heterodimer (MutLα). Co-immunoprecipitation analyses revealed that MutLα, and also MSH2 and MSH6, components of the MutSα heterodimer, form complexes with Polη in human cells. Although MutSα had been reported to interact with C-terminal residues of Polη, MutLα and MutSα co-precipitated with C-terminally truncated Polη, suggesting that MutSα can interact with Polη through MutLα. MMR proteins were more abundant in the Polη complex on the chromatin of S phase-synchronized cells than of asynchronous cells, suggesting that the interaction between Polη and MLH1 is involved in DNA replication.     

Site-specific protein modification to identify the MutL interface of MutH

We have mapped the region for the protein interaction site of the Escherichia coli mismatch repair protein MutH for its activator protein MutL by a site-specific protein modification approach. For this purpose we generated a cysteine-free variant of MutH and 12 variants thereof, each containing a single cysteine residue at surface positions selected on the basis of available structural and sequence information for MutH. All MutH variants displayed wild type activity both in vivo and in vitro. These variants were then site-specifically modified at their cysteine residues with thiol-specific reagents and then tested for their ability to be stimulated in their DNA cleavage activity by the activator protein MutL. Thereby we were able to identify a defined region in the MutH protein that is important for interaction with MutL, and most likely represents the MutL binding site of MutH.     

Resolving Holliday junctions with Escherichia coli UvrD helicase

The Escherichia coli UvrD helicase is known to function in the mismatch repair and nucleotide excision repair pathways and has also been suggested to have roles in recombination and replication restart. The primary intermediate DNA structure in these two processes is the Holliday junction. UvrD has been shown to unwind a variety of substrates including partial duplex DNA, nicked DNA, forked DNA structures, blunt duplex DNA and RNA-DNA hybrids. Here, we demonstrate that UvrD also catalyzes the robust unwinding of Holliday junction substrates. To characterize this unwinding reaction we have employed steady-state helicase assays, pre-steady-state rapid quench helicase assays, DNaseI footprinting, and electron microscopy. We conclude that UvrD binds initially to the junction compared with binding one of the blunt ends of the four-way junction to initiate unwinding and resolves the synthetic substrate into two double-stranded fork structures. We suggest that UvrD, along with its mismatch repair partners, MutS and MutL, may utilize its ability to unwind Holliday junctions directly in the prevention of homeologous recombination. UvrD may also be involved in the resolution of stalled replication forks by unwinding the Holliday junction intermediate to allow bypass of the blockage.     

Mutations in the MutSalpha interaction interface of MLH1 can abolish DNA mismatch repair

MutLα, a heterodimer of MLH1 and PMS2, plays a central role in human DNA mismatch repair. It interacts ATP-dependently with the mismatch detector MutSα and assembles and controls further repair enzymes. We tested if the interaction of MutLα with DNA-bound MutSα is impaired by cancer-associated mutations in MLH1, and identified one mutation (Ala128Pro) which abolished interaction as well as mismatch repair activity. Further examinations revealed three more residues whose mutation interfered with interaction. Homology modelling of MLH1 showed that all residues clustered in a small accessible surface patch, suggesting that the major interaction interface of MutLα for MutSα is located on the edge of an extensive β-sheet that backs the MLH1 ATP binding pocket. Bioinformatic analysis confirmed that this patch corresponds to a conserved potential protein–protein interaction interface which is present in both human MLH1 and its E.coli homologue MutL. MutL could be site-specifically crosslinked to MutS from this patch, confirming that the bacterial MutL–MutS complex is established by the corresponding interface in MutL. This is the first study that identifies the conserved major MutLα–MutSα interaction interface in MLH1 and demonstrates that mutations in this interface can affect interaction and mismatch repair, and thereby can also contribute to cancer development.