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- W43918703 abstract "The timely initiation of DNA replication is achieved through a process calledlicensing, which ensures that only after passage through mitosis the chromatinbecomes competent for a new round of DNA replication. Licensing involves thestepwise formation of a multiprotein complex at the origins of replication, called prereplicativecomplex. A major component of this complex is Cdt1, which is also acritical and evolutionarily conserved proteolytic target of the DNA damagecheckpoint. In metazoans, a small protein called Geminin binds to Cdt1, inhibitingreplication licensing. Geminin has been proposed to coordinate cell cycle anddifferentiation events through balanced interactions with the cell cycle regulator Cdt1and with homeobox transcription factors and chromatin remodeling activitiesimplicated in cell fate decisions.Here we show that Geminin is cleaved in primary cells and cancer cell linesinduced to undergo apoptosis by a variety of stimuli. Geminin targeting is mediatedby caspase-3 both in vivo and in vitro. Two sites at the carboxyl terminus of Gemininare cleaved by the caspase (termed K1 and K2), producing truncated forms ofGeminin. We provide evidence that Geminin cleavage is regulated by phosphorylationby Casein kinase II. Geminin cleaved at site K1 has a proapoptotic effect. Geminincleaved at the site K2 has lost the ability to interact with Brahma (Brm), a catalyticsubunit of the SWI/SNF chromatin remodeling complex, while binding efficiently toCdt1, indicating that targeting of Geminin during apoptosis differentially affectsinteractions with its binding partners.In the second part of this thesis, we investigated the spatiotemporal regulation ofCdt1 in DNA-damaged cells. We introduced and characterized a method of inducinglocalized DNA damage, based on a UVA-pulsed laser. Using this method, we showthat Cdt1 is recruited at the site of damage in parallel to the recruitment of knownresponse factors, such as γH2AX, BRCA1, MRE11, Ku70 etc. Cdt1 recruitment at thesite of damage precedes its degradation in both cancer cell lines and primary cells.Mutated forms of Cdt1 and siRNA for Cdt1-interacting proteins show that Cdt1accumulation at the site of damage requires interactions with the protein PCNA. Inaddition, we found that Cdt2, a member of the Cul4-DDB1Cdt2 complex thatubiquitinates Cdt1 after DNA damage, is recruited at the site of damage. Experimentsof knocking down the protein expression using siRNA and quantification of the recruitment kinetics address the molecular interplay of these proteins for therecruitment at the site of damage, while FRAP experiments revealed their dynamics.Taken together our results suggest that following DNA damage, PCNA is recruited tothe site of damage, and acts as a scaffold for the dynamic interaction of Cdt1 with thedamaged sites. The accumulation of Cdt1 at the site of damage suggest a possibleinvolvement this cell cycle regulator in the DNA damage response." @default.
- W43918703 created "2016-06-24" @default.
- W43918703 creator A5050700908 @default.
- W43918703 date "2021-08-31" @default.
- W43918703 modified "2023-10-18" @default.
- W43918703 title "Διερεύνηση της ρύθμισης των πρωτεϊνών του κυτταρικού κύκλου Cdt1 και Geminin σε κύτταρα με βλάβες στο DNA και σε αποπτωτικά κύτταρα" @default.
- W43918703 doi "https://doi.org/10.12681/eadd/27711" @default.
- W43918703 hasPublicationYear "2021" @default.
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