The replisome is a dynamic multi-protein molecular machine that synthesizes DNA allowing for the passage of genetic information from one generation to another. Intrinsic dynamics and complexity of the replisome make it challenging for structural and biochemical studies using ensemble-averaging techniques. As a consequence, to date structural information is scarce and the molecular mechanisms for coordination of DNA synthesis are poorly understood.
Nearly three hundred mutations have been identified to date in the sequence of mitochondrial replication proteins. These mutations play a causative role in development of cancer and neurodegenerative mitochondrial disorders. Due to the lack of structural data, an exact mechanism by which altered replication proteins affect replisome function is not known.
Our laboratory integrates structural approaches, in particular single-particle cryo-electron microscopy (cryo-EM) and single-molecule methods to study DNA replication and repair in human mitochondria. We also develop novel correlative light and electron microscopy (CLEM) techniques that allow simultaneous visualization of an enzymatic activity and structure determination. By using the above methods we expect to reveal mechanistic information that may subsequently facilitate development of novel therapeutics.
Kulczyk, A.W.*, Moeller, A., Meyer P., Sliz, P., Richardson, C.C*. (2017). Cryo-EM structure of the replisome reveals multiple interactions coordinating DNA synthesis. Proceedings of the National Academy of Sciences of the USA, 114, 1848-1856.
Kulczyk, A.W., Richardson, C.C. (2016). The Replication System of Bacteriophage T7. Book chapter in The Enzymes, 39, 89-136.
Geertsema, H.J., Kulczyk, A.W., Richardson, C.C., van Oijen A. (2014). Single-molecule studies of polymerase dynamics and stoichiometry at the bacteriophage T7 replication machinery. Proceedings of the National Academy of Sciences of the USA, 111, 4073-4078.
Kulczyk, A.W.*, Richardson, C.C. (2012). Molecular interactions in bacteriophage T7 priming complex. Proceedings of the National Academy of Sciences of the USA, 109, 9408-9413.
Kulczyk, A.W., Akabayov, B., Lee, S-J. et al. (2012). An interaction between DNA polymerase and helicase is essential for the high processivity of the replisome. Journal of Biological Chemistry, 287(46), 39050-39060.
Loparo, J., Kulczyk, A.W., Richardson, C.C., van Oijen A. (2011). Observing polymerase exchange by simultaneous measurements of replisome structure and function at the single-molecule level. Proceedings of the National Academy of Sciences of the USA, 108, 3584-3589.
Kulczyk, A.W., Tanner N., Loparo J., Richardson C., van Oijen A. (2010). Direct observation of enzymes replicating DNA using a single-molecule DNA stretching assay. Journal of Visual Experiments, 37, id: 1689, doi: 10.3791/1689.
Kulczyk, A.W., Yang, J., Neuhaus, D. (2004). Solution structure and DNA binding of the zinc finger domain from DNA ligase IIIa. Journal of Molecular Biology, 341, 723-738.