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Peter Friedhoff

Publications and source records attributed to Peter Friedhoff.

13 recordsLinked to original sources
PubMed2007

Engineering site-specific endonucleases.

Site-specific endonucleases are involved in many aspects of the biochemistry of nucleic acids. Restriction enzymes and their relatives have become paradigms for enzymes acting on DNA. Numerous efforts have been devoted to changing their specificity by rational protein design, with, by and large, little success, presumably because the recognition process is highly redundant and recognition and catalysis are tightly coupled. This chapter describes one of the few successful examples of a change in specificity--namely the conversion of the mismatch repair nuclease, MutH, which, when stimulated by MutS and MutL, nicks hemimethylated d(GATC) sites, into a variant that cleaves fully methylated DNA as well as hemimethylated and unmethylated DNA. This chapter will describe the various steps involved in this design project, starting from the analysis of the structure and the identification of candidate amino acid residues responsible for sensing the methylation status, to the generation and characterization of MutH variants with an altered specificity toward hemimethylated d(GATC) sites.

Amino Acid Sequence↗
PubMed2006

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

MutLalpha, a heterodimer of MLH1 and PMS2, plays a central role in human DNA mismatch repair. It interacts ATP-dependently with the mismatch detector MutSalpha and assembles and controls further repair enzymes. We tested if the interaction of MutLalpha with DNA-bound MutSalpha 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 MutLalpha for MutSalpha is located on the edge of an extensive beta-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 MutLalpha-MutSalpha 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.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Structural and functional analysis of the MutS C-terminal tetramerization domain.

The Escherichia coli DNA mismatch repair (MMR) protein MutS is essential for the correction of DNA replication errors. In vitro, MutS exists in a dimer/tetramer equilibrium that is converted into a monomer/dimer equilibrium upon deletion of the C-terminal 53 amino acids. In vivo and in vitro data have shown that this C-terminal domain (CTD, residues 801-853) is critical for tetramerization and the function of MutS in MMR and anti-recombination. We report the expression, purification and analysis of the E.coli MutS-CTD. Secondary structure prediction and circular dichroism suggest that the CTD is folded, with an alpha-helical content of 30%. Based on sedimentation equilibrium and velocity analyses, MutS-CTD forms a tetramer of asymmetric shape. A single point mutation (D835R) abolishes tetramerization but not dimerization of both MutS-CTD and full-length MutS. Interestingly, the in vivo and in vitro MMR activity of MutS(CF/D835R) is diminished to a similar extent as a truncated MutS variant (MutS800, residues 1-800), which lacks the CTD. Moreover, the dimer-forming MutS(CF/D835R) has comparable DNA binding affinity with the tetramer-forming MutS, but is impaired in mismatch-dependent activation of MutH. Our data support the hypothesis that tetramerization of MutS is important but not essential for MutS function in MMR.

Amino Acid Sequence↗
PubMed2006

Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry.

To investigate protein-protein interaction sites in the DNA mismatch repair system we developed a crosslinking/mass spectrometry technique employing a commercially available trifunctional crosslinker with a thiol-specific methanethiosulfonate group, a photoactivatable benzophenone moiety and a biotin affinity tag. The XACM approach combines photocrosslinking (X), in-solution digestion of the crosslinked mixtures, affinity purification via the biotin handle (A), chemical coding of the crosslinked products (C) followed by MALDI-TOF mass spectrometry (M). We illustrate the feasibility of the method using a single-cysteine variant of the homodimeric DNA mismatch repair protein MutL. Moreover, we successfully applied this method to identify the photocrosslink formed between the single-cysteine MutH variant A223C, labeled with the trifunctional crosslinker in the C-terminal helix and its activator protein MutL. The identified crosslinked MutL-peptide maps to a conserved surface patch of the MutL C-terminal dimerization domain. These observations are substantiated by additional mutational and chemical crosslinking studies. Our results shed light on the potential structures of the MutL holoenzyme and the MutH-MutL-DNA complex.

Adenosine Triphosphatases↗
PubMed2005

Analysis of the quaternary structure of the MutL C-terminal domain.

The dimeric DNA mismatch repair protein MutL has a key function in communicating mismatch recognition by MutS to downstream repair processes. Dimerization of MutL is mediated by the C-terminal domain, while activity of the protein is modulated by the ATP-dependent dimerization of the highly conserved N-terminal domain. Recently, a crystal structure analysis of the Escherichia coli MutL C-terminal dimerization domain has been reported and a model for the biological dimer was proposed. In this model, dimerization is mediated by the internal (In) subdomain comprising residues 475-569. Here, we report a computational analysis of all protein interfaces observed in the crystal structure and suggest that the biological dimer interface is formed by a hydrophobic surface patch of the external (Ex) subdomain (residues 432-474 and 570-615). Moreover, sequence analysis revealed that this surface patch is conserved among the MutL proteins. To test this hypothesis, single and double-cysteine variants of MutL were generated and tested for their ability to be cross-linked with chemical cross-linkers of various size. Finally, deletion of the C-terminal residues 605-615 abolished homodimerization. The biochemical data are fully compatible with a revised model for the biological dimer, which has important implications for understanding the heterodimerization of eukaryotic MutL homologues, modeling the MutL holoenzyme and predicting protein-protein interaction sites.

Adenosine Triphosphatases↗
PubMed2004

Mapping protein-protein interactions between MutL and MutH by cross-linking.

Strand discrimination in Escherichia coli DNA mismatch repair requires the activation of the endonuclease MutH by MutL. There is evidence that MutH binds to the N-terminal domain of MutL in an ATP-dependent manner; however, the interaction sites and the molecular mechanism of MutH activation have not yet been determined. We used a combination of site-directed mutagenesis and site-specific cross-linking to identify protein interaction sites between the proteins MutH and MutL. Unique cysteine residues were introduced in cysteine-free variants of MutH and MutL. The introduced cysteines were modified with the cross-linking reagent 4-maleimidobenzophenone. Photoactivation resulted in cross-links verified by mass spectrometry of some of the single cysteine variants to their respective Cys-free partner proteins. Moreover, we mapped the site of interaction by cross-linking different combinations of single cysteine MutH and MutL variants with thiol-specific homobifunctional cross-linkers of varying length. These results were used to model the MutH.MutL complex and to explain the ATP dependence of this interaction.

Adenosine Triphosphatases↗
PubMed2003

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.

Adenosine Triphosphatases↗
PubMed2003

Tyr212: a key residue involved in strand discrimination by the DNA mismatch repair endonuclease MutH.

The molecular mechanism of how the dam-methylation status of the DNA is recognized during DNA mismatch repair by the strand discrimination endonuclease MutH is not known. A comparison of the crystal structure of MutH with those of co-crystal structures of several restriction endonucleases, together with a multiple sequence alignment of MutH and related proteins suggested that Phe94, Arg184 and Tyr212 could be involved in discrimination between a methylated or unmethylated adenine in the d(GATC) sequence. A mutational analysis revealed that the variants R184A and Y212S, but not F94A, were substantially reduced in their ability to complement a mismatch repair deficiency in a mutH(-) Escherichia coli strain. In vitro, R184A displayed a strongly reduced endonuclease activity, whereas the Y212S variant has almost completely lost its preference for cleaving the unmethylated strand at hemimethylated d(GATC) sites. Furthermore, the Y212 variant can cleave fully methlyated d(GATC) sites at a comparable rate to unmethylated d(GATC) sites. This demonstrates that Tyr212 is an important, if not the only amino acid residue in MutH for sensing the methylation status of the DNA.

Amino Acid Sequence↗
PubMed2002

Haemophilus influenzae and Vibrio cholerae genes for mutH are able to fully complement a mutH defect in Escherichia coli.

MutH, MutL and MutS are essential components of the mismatch repair system in Escherichia coli. Whereas mutS and mutL genes are found in most organisms, the mutH gene is limited to some proteobacteria. We show here that the cloned genes of MutH from Vibrio cholerae and Haemophilus influenzae are able to fully complement a mutH defect in E. coli. Moreover, the purified proteins were shown to be dam methylation sensitive endonucleases, which can be activated by the E. coli MutL protein. These results allow to narrow down regions that are important for the interaction of MutH with MutL.

Adenosine Triphosphatases↗
PubMed2002

Evolutionary relationship between different subgroups of restriction endonucleases.

The type II restriction endonuclease SsoII shows sequence similarity with 10 other restriction endonucleases, among them the type IIE restriction endonuclease EcoRII, which requires binding to an effector site for efficient DNA cleavage, and the type IIF restriction endonuclease NgoMIV, which is active as a homotetramer and cleaves DNA with two recognition sites in a concerted reaction. We show here that SsoII is an orthodox type II enzyme, which is active as a homodimer and does not require activation by binding to an effector site. Nevertheless, it shares with EcoRII and NgoMIV a very similar DNA-binding site and catalytic center as shown here by a mutational analysis, indicative of an evolutionary relationship between these three enzymes. We suggest that a similar relationship exists between other orthodox type II, type IIE, and type IIF restriction endonucleases. This may explain why similarities may be more pronounced between members of different subtypes of restriction enzymes than among the members of a given subtype.

Amino Acid Motifs↗
PubMed2002

An efficient method for the preparation of long heteroduplex DNA as substrate for mismatch repair by the Escherichia coli MutHLS system.

We present a method that allows preparing long DNA containing defined mismatches without the use of gel electrophoretic or chromatographic purification steps. The preparation starts with the synthesis of two PCR products, which are identical except for those positions that will later form the mismatches. One of the PCR primers must be 5'-phosphorylated, such that in two separate reactions two PCR products are obtained, which are 5'-phosphorylated in one strand. After removal of the phosphorylated strands by lambda-exonuclease, the resulting single strands are hybridized to form the mismatch-containing heteroduplex. The application of this procedure is demonstrated for the analysis of the Escherichia coli MutHLS system.

Adenosine Triphosphatases↗
PubMedDate not supplied

Application of the C4'-alkylated deoxyribose primer system (CAPS) in allele-specific real-time PCR for increased selectivity in discrimination of single nucleotide sequence variants.

This study describes a quantitative real-time PCR-based approach for discrimination of single nucleotide sequence variants, called CAPS (C4' alkylated primer system). To increase the discrimination potential of DNA polymerases against competing sequence variants of single nucleotides, 3'-terminally modified primers were designed carrying a methyl residue bound to the C4' of the thymidylate deoxyribose. In a model sequence system positional dependencies of modified thymidylate (at -1, -2, -3) were tested for their influence on discrimination. Highest discrimination factors were obtained with the modification at the ultimate 3'-position. In a comparison between Taq and Pwo DNA polymerases, substantial better results were obtained by Taq DNA polymerase. In contrast to conventional PCR methods for discrimination of sequence variants, achieving a maximum discrimination potential of about 20, CAPS is capable of obtaining sequence-specific amplifications of a desired target among discriminated templates with a dynamic range of 1:100. Therefore, CAPS is a method able to quantitatively discriminate two sequence variants only differing in a single base (e.g., SNP alleles or point mutations). The range of applications of this easy to perform, fast and reliable technique reaches from medical diagnostics, transplantation medicine, molecular and cell biology to human genetics. Targeting of SNPs assures a universal exertion of this method, since these markers are gender-independent, highly abundant and ubiquitous.

Alkylation↗