Protein labelling and fluorescence FRET experiments for exploring the conformational dynamics in the regulation of the DNA repair protein APE1.

Natural Sciences
Spain
Leioa (near Bilbao)
Period: 
1 Sep, 2020 to 31 May, 2021
Deadline: 
1 Nov, 2020

General information

Duration: 
8 months
Commitment: 
3-4 days per week
Description: 

APE1 (apurinic apyrimidinic endonuclease 1) is a key factor in one of the DNA repair pathways, known as “Base Excision Repair” (BER), which activates upon DNA “lesions” elicited by oxidative damage, among others. APE1 recognizes “abasic” sites in the DNA, where the base has been lost either due to spontaneous depurination, or by the action of a repair glycosidase, and hydrolizes the 5´phosphodiester link. APE1 has been deeply characterized at structural and catalytical levels, but less is known about its regulation, which could be mediated by interactions with other proteins. APE1 interacts with nucleophosmin (NPM1), an abundant nucleolar protein with several functions affecting cell homeostasis. NPM1 is implicated in the cellular stress response and in DNA repair, but its involvement in such processes, in particular, how could it participate together with APE1 in the BER route, has not been yet elucidated. We plan to explore the conformational changes related with APE1 activity on DNA, and how could these be modulated by NPM1. To that aim, we have designed FRET (Förster resonance energy transfer) experiments to monitor the comformational rearrangements that may take place in APE1 as a result of its DNA binding, catalysis and interaction with NPM1. In particular, the putatively regulatory role of the ca. 40 residues long APE1 N-terminal region, conformationally flexible and therefore missing from the crystallographic models, should be analyzed. After labelling APE1 with different fluorphores and in different positions, these assays will be performed firstly in “bulk” and on their basis, further single-molecule type experiments, of higher spatio-temporal resolution will be scheduled.

Compensation: 
No financial compensation
Years of Experience required: 
No

Keywords

APE1  DNA repair  protein conformation  fluorescence spectroscopy  single-molecule  
Requirements
Languages: 
English: Independent User B2
Level of Studies: 
Master