DNA mismatch repair Lab

Department                        Molecular Genetics

Principal investigator     Joyce Lebbink

E-mail address                   j.lebbink@erasmusmc.nl

Website                               https://www.erasmusmc.nl/en/research/researchers/lebbink-joyce

Molecular mechanism underlying mismatch specificity of MutS during DNA mismatch repair

DNA mismatch repair (MMR) maintains genome stability by repairing DNA replication errors such as DNA mismatches. In E. coli, MMR is initiated by MutS, MutL and MutH. MutS recognizes the mismatch and upon binding of ATP, forms a sliding clamp which recruits MutL, leading to MutH activation and DNA strand incision at a hemimethylated GATC site. To understand how MutS attains mismatch specificity, we create mutations at the interface of its mismatch binding and connector domain. These MutS variants loose mismatch specificity and activate MMR on DNA without errors. Using biochemical and biophysical approaches we will characterize the conformational transitions in wild type and mutant MutS, to understand the molecular mechanism underlying mismatch specificity.

Further reading (click to link to article)

Fernandez-Leiro et. al.  (2021). The selection process of licensing a DNA mismatch for repair. NATURE STRUCTRAL & MOLECULAR BIOLOGY. VOL 28 , 373-381.

Coordination of mismatch repair by MutSα ATPase activity

The DNA mismatch repair (MMR) system corrects mismatches and small insertion/deletion loops that arise during DNA replication. In humans, MMR is initiated when the MutSα complex recognizes a DNA mismatch in the newly replicated DNA. This complex recruit MutLα, which contains the endonuclease activity and coordinates the incision of the error-containing strand, followed by excision of the error and resynthesis and ligation to restore the correct DNA sequence.
Mutations affecting critical residues in the ATPase domain of MutSα from yeast have been shown to impair ATP binding, hydrolysis and sliding clamp formation, which are essential steps for effective mismatch recognition and repair. These residues are conserved in the human homolog. In this project we will construct ATPase mutants in human MutSα and use biochemical approaches to study their effect on the different MMR reactions steps.

Further reading (click to link to article)

Hess, M. T., Mendillo, M. L., Mazur, D. J., & Kolodner, R. D. (2006). Biochemical basis for dominant mutations in the Saccharomyces cerevisiae MSH6 gene. Proceedings of the National Academy of Sciences of the United States of America, 103(3), 558–563. https://doi.org/10.1073/pnas.0510078103

Understanding the effect of MutLα mutations on human DNA Mismatch Repair

The DNA mismatch repair (MMR) system corrects mismatches and small insertion/deletion loops that arise during DNA replication. In humans, MMR is initiated when the MutSα complex recognizes a DNA mismatch in the newly replicated DNA. This complex recruit MutLα (composed of MLH1 and PMS2 subunits), which contains the endonuclease activity and coordinates the incision of the error-containing strand, followed by excision of the error and resynthesis and ligation to restore the correct DNA sequence.
Loss-of-function mutations in MMR proteins result in the accumulation of mutations throughout the genome and are a major cause of Lynch syndrome (hereditary nonpolyposis colorectal cancer), which predisposes to colorectal and other cancers. In this project we will characterize some of these disease-associated variants, particularly in the MLH1 and PMS2 subunits of MutLα, possibly disrupting its endonuclease function or impair its interaction with other MMR proteins. Using biochemically reconstituted MMR assays, we aim to learn how these residues contribute to the molecular mechanism of mismatch repair.

Further reading (click to link to article)

Wolf, K., Kosinski, J., Gibson, T. J., Wesch, N., Dötsch, V., Genuardi, M., Cordisco, E. L., Zeuzem, S., Brieger, A., & Plotz, G. (2023). A conserved motif in the disordered linker of human MLH1 is vital for DNA mismatch repair and its function is diminished by a cancer family mutation. Nucleic acids research, 51(12), 6307–6320. https://doi.org/10.1093/nar/gkad418

Roles of Canonical and Alternative RPA in DNA Mismatch Repair

Replication Protein A (RPA), composed of the subunits RPA1, RPA2, and RPA3, is a key player in mismatch repair. Beyond its essential role in DNA replication, RPA can melt unusual DNA structures and modulate nuclease activities. In primates, there is an alternative isoform known as Alternative-RPA (Alt-RPA), which consists of RPA1, RPA3, and a primate-specific RPA4 subunit replacing RPA2. Interestingly, both RPA and Alt-RPA are upregulated in Huntington’s disease, a disorder caused by expansions of CAG trinucleotide repeats.
Preliminary in vitro experiments using RPA-depleted extracts suggest that low levels of either RPA or Alt-RPA can enhance GT mismatch repair. However, increasing RPA levels further stimulates GT repair, while increasing Alt-RPA has the opposite effect, reducing mismatch repair efficiency. Together, these observations suggest that RPA and Alt-RPA may differentially regulate MMR activity. In this project we aim to characterize at which reaction steps this antagonistic regulation takes place.

Further reading (click to link to article)

Gall-Duncan, T., Luo, J., Jurkovic, C. M., Fischer, L. A., Fujita, K., Deshmukh, A. L., Harding, R. J., Tran, S., Mehkary, M., Li, V., Leib, D. E., Chen, R., Tanaka, H., Mason, A. G., Lévesque, D., Khan, M., Razzaghi, M., Prasolava, T., Lanni, S., Sato, N., … Pearson, C. E. (2023). Antagonistic roles of canonical and Alternative-RPA in disease-associated tandem CAG repeat instability. Cell, 186(22), 4898–4919.e25. https://doi.org/10.1016/j.cell.2023.09.008