State-of-the-art and preparatory work

The DNA mismatch repair system (MMR) is involved in several cellular processes including repair of replication errors but also anti‐recombination and DNA damage signalling. The structure of the principal MMR proteins MutS and MutL (and MutH in Escherichia coli) are known; however, little is known about the structure of the complex that is coordinating the action of the MMR machinery. In the past the focus has been on the process of mismatch recognition by MutS and on the role of its ATPase (1). Much less is known about the function of MutL which is structurally homologous to DNA topoisomerases and Hsp90‐related proteins. The N‐terminal domain, which harbours an ATPase centre and the binding site for MutS, is connected by a flexible linker to a C‐terminal domain containing additional functional sites of ill defined function in MMR (2,3). Recently, we reported the physical and functional connection between MutL and other repair pathways (4). We have established for the E. coli MMR proteins a system to trap and monitor the formation of the proteincomplexes and conformational changes using chemical cross‐linking and fluorescence resonance energy transfer (FRET). Notably, interactions of MutL with partners other than MutS are mediated by both the N‐ and C‐terminal domain (5,6). Moreover, we identified a regulatory Zn‐binding site in bacterial and human MutL, not present in E. coli MutL, which is crucial for the communication between the ATPase and the C‐terminal domain and MutL’s function in MMR (2‐7). Importantly, the combination of click chemistry and genetically encoded unnatural amino acids will enable us to sitespecifically modify MMR proteins, e.g. from humans, that contain essential cysteine residues.

References

1. Zhai, J. & Hingorani, M. M. (2010) Saccharomyces cerevisiae Msh2‐Msh6 DNA binding kinetics reveal a mechanism of targeting sites for DNA mismatch repair. Proc Natl Acad Sci U S A 107, 680‐685.

2. Kosinski, J., Plotz, G., Guarne, A., Bujnicki, J. M. & Friedhoff, P. (2008) The PMS2 subunit of human MutLalpha contains a metal ion binding domain of the iron‐dependent repressor protein family. J Mol Biol 382, 610‐627.

3. Duppatla, V., Bodda, C., Urbanke, C., Friedhoff, P. & Rao, D. N. (2009) The C‐terminal domain is sufficient for endonuclease activity of Neisseria gonorrhoeae MutL. Biochem J 423, 265‐277.

4. Heinze, R. J., Giron‐Monzon, L., Solovyova, A., Elliot, S. L., Geisler, S., Cupples, C. G., Connolly, B. A. & Friedhoff, P. (2009) Physical and functional interactions between Escherichia coli MutL and the Vsr repair endonuclease. Nucleic Acids Res 37, 4453‐4463.

5. Ahrends, R., Kosinski, J., Kirsch, D., Manelyte, L., Giron‐Monzon, L., Hummerich, L., Schulz, O., Spengler, B. & Friedhoff, P. (2006) Identifying an interactio site between MutH and the C‐terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry. Nucleic Acids Res 34, 3169‐3180.

6. Giron‐Monzon, L., Manelyte, L., Ahrends, R., Kirsch, D., Spengler, B. & Friedhoff, P. (2004) Mapping protein‐protein interactions between MutL and MutH by cross‐linking. J Biol Chem 279, 49338‐49345.

7. Pillon, M. C., Lorenowicz, J. J., Uckelmann, M., Klocko, A. D., Mitchell, R. R., Chung, Y. S., Modrich, P., Walker, G. C., Simmons, L. A., Friedhoff, P. & Guarné, A. (2010) Structure of the endonuclease domain of MutL: unlicenced to cut ‐ MOLECULAR‐CELL‐S‐10‐00080 accepted.

8. Kaya E, Gutsmiedl, K., Vrabel, M., Muller, M., Thumbs, P. & Carell, T. (2009) Synthesis of threefold glycosylated proteins using click chemistry and genetically encoded unnatural amino acids. Chembiochem 10:2858‐2861.

Aims

The overall aim of this project is to understand the molecular structure and mechanism of how the MutS‐MutL complex responds to DNA mismatches and damages. Aim

I. is to determine the structure of the dynamic complex between MutS, MutL and DNA from E. coli, B. subtilis and humans. II. is to test the communication between the N‐terminal ATPase and the C‐terminal domain of MutL from E. coli, B. subtilis and humans and its interaction with DNA. III. is to identify the interaction partners of MutL in Rhodobacter and Bacillus species.

IV. is to address the role of chromatin structure on the eukaryotic MMR process.

Work programme and methods

For Aim I, we will start with the chemical trapping of the E. coli MutS‐MutL‐DNA complex for subsequent structural analysis (e.g. SAXS, SFM or X‐ray crystallography in collaboration with Prof. T. Sixma, Amsterdam) and then transfer this approach to the B. subtilis and the human system by exploiting the knowledge obtained from the E. coli system. Aim II: The method of choice for studying conformational changes in the MutL proteins (E. coli, B. subtilis and human) in response to ATP binding and DNA damage will be FRET (down to the single‐molecule level) using site‐specifically labelled proteins. Cysteine residues or unnatural amino acid residues will be incorporated at strategic positions to allow site‐specific labelling using maleimide or click‐chemistry, respectively. Aim III: In order to identify interaction partners of MutL in response to specific DNA mismatches and various types of DNA damage, we will use a combination of cross‐linking, affinity purification (using affinitytagged DNA or protein) and mass spectrometry analysis and again taking advantage from our experience with the E. coli system. Hits will be validated by biophysical and functional interaction analysis. For Aim IV we will exploit the experience of the groups working on chromatin structure and remodelling to generate suitable DNA heteroduplex substrates for the analysis of eukaryotic MMR reactions.

Titles for dissertations (prospective)

• Structure of the MutS‐MutL‐DNA complex

• Dissection of the ATP‐induced conformational cycle involving the molecular matchmaker MutL

• Identification of MutL interaction partners in response to DNA damage

• The role of chromatin structure on MMR

Relationships/connections within the research training group

Kubareva/Oretskaya: Investigation of nucleic acids‐protein and protein‐protein interactions taking part in gene expression regulation in bacterial cells using modified oligonucleotides

Klug: Role of MMR in oxidative DNA damage response in R. sphaeroides

Hartmann: Reverse genetics in B. subtitlis/generation of MMR knockouts in B. subtilis

Pingoud, Wende: Facilitated diffusion to locate specific target site on the DNA

Renkawitz, Brehm: Chromatin structure and remodelling

Benefits of the scientific exchange

The project will have several benefits. (1) Complementary methodologies present in the other groups can be learned (e.g. Reverse genetics in bacteria other than E. coli) or taught (e.g. protein crosslinking /modification). (2) Specific reagents/materials will be exchanged (e.g. modified DNA substrates, chromatin). (3) Students will learn to engage in team‐work in joint projects (e.g. response to oxidative DNA damage in Rhodobacter). (4) Students will take advantage of existing networks of the partners (e.g. single‐molecule analysis by Pingoud and Wende with P. Desbiolles, Paris).