Petr Cejka si è laureato nel 2000 presso l’Università “Charles University” di Praga, ed ha conseguito il dottorato di ricerca nel 2004 presso l’Università di Zurigo. Durante i suoi studi di dottorato con il Prof. J. Jiricny, Petr Cejka ha studiato i meccanismi di riparazione del DNA in cellule umane. In particolare, ha studiato come la riparazione dei “mismatches” nel DNA possa influenzare la sensibilità del DNA verso gli agenti metilanti che vengono usati per la terapia antitumorale. Petr Cejka ha in seguito ottenuto una borsa di studio dal Fondo Nazionale Svizzero ed è entrato a far parte del gruppo del Prof. S. Kowalczykowski presso l’University of California, Davis, USA. Durante gli anni di post dottorato il Dott. Cejka si è specializzato nell’utilizzo di tecniche di biochimica delle proteine ed i suoi studi hanno contribuito alla comprensione dei meccanismi di ricombinazione omologa del DNA. Nel 2011 ha ottenuto dal Fondo Nazionale Svizzero una posizione di professore associato ed è tornato all’Università di Zurigo, presso l'”Institute of Molecular and Cancer Research” dove ha avviato un suo gruppo di ricerca indipendente. Il Prof. Cejka ha ricevuto il “Dr. Ernst Th. Jucker Award 2015” per il suo contributo alla ricerca sul cancro e il “Friedrich Miescher Award 2017” per i suoi risultati nel campo della biochimica. Nel 2016 il Prof. Cejka si è trasferito all’ IRB come direttore di laboratorio ed ha in seguito ottenuto una posizione di professore associato all’USI. Il Prof. Cejka è interessato alla comprensione dei meccanismi che le cellule mettono in atto per riparare il DNA danneggiato, con particolare attenzione ad un meccanismo di riparazione chiamato Ricombinazione Omologa. Il Prof. Cejka ha ottenuto un ERC (European Research Council) consolidator grant nel 2016 e un ERC advanced grant nel 2021.
First steps in homologous recombination: DNA end resection
Homologous recombination is initiated by the nucleolytic degradation (resection) of the 5'-terminated DNA strand of the DNA break. This leads to the formation of 3'-tailed DNA, which becomes a substrate for the strand exchange protein RAD51 and primes DNA synthesis during the downstream events in the recombination pathway. DNA end resection thus represents a key process that commits the repair of DNA breaks into recombination. Research from multiple laboratories established that DNA end resection is in most cases a two-step process. It is initiated by the nucleolytic degradation of DNA that is at first limited to the vicinity of the broken DNA end. This is carried out by the Mre11-Rad50-Xrs2 (MRX) complex and Sae2 proteins in yeast, and MRE11-RAD50-NBS1 (MRN) and CtIP proteins in human cells. We could reconstitute these reactions in vitro, and demonstrated that Sae2 and CtIP stimulate a cryptic endonuclease activity within the yeast MRX or human MRN complex, respectively. The activity of Sae2/CtIP is absolutely dependent on its phosphorylation. The reconstituted DNA clipping reaction allows us to investigate the mechanism of this process as well as its regulation by posttranslational modifications and additional protein co-factors.
Downstream of MRX-Sae2 and MRN-CtIP, which process only a limited length of DNA, DNA end resection is further catalyzed by Sgs1-Dna2 or Exo1 in yeast and BLM-DNA2, WRN-DNA2 or EXO1 in human cells. We are interested how the functions of these factors integrate in protein complexes to form molecular machines that are uniquely capable to resect long lengths of DNA, which is required for homologous recombination. We are specifically interested in the Dna2 enzyme, and could show that both yeast Dna2 and human DNA2 possess a cryptic helicase activity. We now investigate how the motor activity of Dna2 promotes DNA end resection, as well as how it is regulated in cells. Finally, as some of these enzymes are upregulated in various human cancers, we are also searching for small molecules capable to inhibit these pathways.
Promotion of genetic diversity in meiosis: resolution of recombination intermediates
Promotion of genetic diversity is a key function of sexual reproduction. At the molecular level, this is controlled by the homologous recombination machinery, which exchanges (recombines) DNA fragments between the maternal and paternal genomes. During this process, joint molecules form between the 'mum' and 'dad' chromosomes, leading to intermediates termed double Holliday junctions. These joint molecules are then processed in a way that results in the physical exchange of genetic information between the two recombining chromosomes. This so‐called crossover is an integral and essential part of the meiotic cell division. Results from genetic, cell biological and cytological experiments identified the Mlh1‐Mlh3 heterodimer as part of a protein complex that is required for the generation of crossovers during meiotic homologous recombination. However, the mechanism of this reaction is completely unknown. The aim of our research is to analyze the behavior of the purified recombinant Mlh1‐Mlh3 complex as well that of its partners in the processing of double Holliday junctions. We want to show how Mlh1‐Mlh3 can cleave these structures into exclusively crossover recombination products, and therefore explain the molecular mechanism underlying the generation of diversity in meiosis.
So far, we successfully expressed and purified the yeast Mlh1-Mlh3 and human MLH1-MLH3 recombinant proteins into near homogeneity. We could show that the recombinant MutLγ is indeed a nuclease that nicks double‐stranded DNA in the presence of manganese, similarly to the mismatch repair specific MutLα nuclease. MutLγ binds DNA with a high affinity, and shows a marked preference for Holliday junctions, in agreement with its anticipated activity in their processing. Specific DNA recognition has never been observed with any other eukaryotic MutL homologue. Mismatch repair specific MutLα shows no binding preference to mismatched DNA. MutLγ thus represents a new paradigm for the function of the eukaryotic MutL protein family. Unfortunately, to date, we have not seen any activity on joint molecule intermediates (such as Holliday junctions) in the presence of physiological manganese metal cofactor. This will likely require interplay of Mlh1-Mlh3 with other cellular factors (such as Exo1, Msh4-Msh5, etc.), and is the subject of vigorous research in the laboratory at present.
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