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| Author(s): |
Daiane da Rocha Soares
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) |
| Defense date: | 2014-04-16 |
| Examining board members: |
Maria Carolina Quartim Barbosa Elias Sabbaga;
Bettina Malnic;
Gerhard Wunderlich
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| Advisor: | Maria Carolina Quartim Barbosa Elias Sabbaga |
| Abstract | |
The pre-replication complex in T.brucei is composed of at Orc1/Cdc6 and MCMs helicases. In a previous paper we showed that TbOrc1/Cdc6 can bind and hydrolyze ATP in vitro. Based on that, the objective of this study is to evaluate the importance of ATP binding and hydrolysis to the formation and stability of the pre - replication complex in T.brucei. For this purpose, T. brucei Orc1/Cdc6 recombinant proteins were generated mutated at regions on Walker A (TbOrc1/Cdc6K79T) and sensor 2 (TbOrc1/Cdc6R251 , 252E) in order to unable the ATP binding and hydrolyzation respectively . Finally , cells expressing TbOrc1/Cdc6K79T or TbOrc1/Cdc6R251 , 252E were evaluated for (i) stability of Orc1/Cdc6 - DNA interaction , (ii) ability to stabilize MCM in DNA , (iii) ability to replicate its DNA . The mutation in the sensor 2 region of T.brucei (TbOrc1/Cdc6R251 , 252E) drastically reduced the ATPase activity compared to the wild-type protein. TbOrc1/Cdc6 mutated in the ATP binding site has lost the ability to interact with ATP (TbOrc1/Cdc6K79T). The overexpression of these genes significantly inhibited cell proliferation causing inefficient loading of MCM DNA and led to failure in cell cycle progression by delaying the phase S. (AU) | |
| FAPESP's process: | 11/05187-6 - Analysis of the importance of ATPase activity of Orc/Cdc6 trypanosomes of the stability of the complex pre DNA replication |
| Grantee: | Daiane da Rocha Soares |
| Support Opportunities: | Scholarships in Brazil - Master |