EVOPRIMPOL · Artificial evolution of a novel multifunctional human polymerase
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2015-05-01 → 2017-04-30
- EU contribution
- €170,122
- Participants
- 1
- Scheme
- MSCA-IF-EF-ST
Lines connect the coordinator with its partners.
Results in brief
Artificial evolution of a novel multifunctional human polymerase
Human PrimPol (HsPP) is a novel primase/polymerase that unlike regular primases, which exclusively incorporate ribonucleotides using DNA as template, PrimPol can incorporate NTPS and preferentially deoxynucleotides (dNTPs) in the presence of a DNA template, either during the priming reaction, or during the elongation stage. Furthermore, analysis performed in vivo showed this enzyme plays an important role in nuclear DNA maintenance, as it is needed for repriming DNA synthesis under stress conditions such as UV irradiation or nucleotide depletion, and its downregulation provokes genome instability. These characteristics convert the study of HsPP in a new and exciting field of research. Apart from the HsPP, very few members of this protein family have been characterized since their discovery 10 years ago. Given that record data belonging to these new class of proteins is limited, election of the protein residues to be changed in order to study their roles in the different activities achieved by HsPP cannot be decided by sequence homology with distant family members, or by comparison of a wild-type (WT) PrimPol with specific mutants associated with human diseases, as is routinely done in the case of regular DNA polymerases. Thus, a methodology able to screen several variants by high-throughput means is needed. Thus, the main objective of the project EVOPRIMPOL, has been the generation of hundreds of variants of HsPP by its randomization and further specific screening. This experimental approach has produced different HsPP variants with enhanced catalytic activities regarding the WT protein. Identification of those amino acids responsible of functional enhancements helps us to understand the structure function relationships of HsPrimPol. Likewise, obtaining evolved HsPPs with modified properties might be exploited for its use in biotechnology, specifically in next generation DNA sequencing and amplification techniques.
Data: CORDIS, © European Union
Project objective
The product of the human gene ccdc111, PrimPol, has been recently identified and characterized by the group of Professor Luis Blanco (CBMSO, Madrid, Spain). This protein is capable of performing both priming and polymerase activities in human cells, contributing to DNA integrity and maintenance during its replication. As an enzyme involved in tolerance to DNA damage, its malfunction gives rise to human diseases. Thus, it might become a target for cancer therapy. In order to exploit the benefits derived from knowledge of PrimPol, a deeper structure-function study must be done. These studies can not be accomplished using standard site-directed mutagenesis protocols and further characterization due to variety activities that this protein displays, the novelty of its discovery and the absence of a solved crystal structure. Thus, EVOPRIMPOL will be a novel, fast and reliable approach to study the role of critical amino acids involved in the different activities shown by the enzyme. This approach is based on the in vitro directed evolution of human gene ccdc111, but unlike other directed studies performed in polymerases, where genes are mutated randomly, in EVOPRIMPOL we will use a new rational design to select those residues that will be randomised; selecting them after the phylogenetic study of all members of the protein superfamily containing PrimPols as well as by homology sequence studies using different DNA polymerases and primases from other superfamilies. Chosen residues will be randomised following an iterative mutagenesis scheme, where evolved variants are subjected to new rounds of mutagenesis mimicking the natural evolution. Thus, improved PrimPols will be obtained with better catalytic properties. Biochemical study of these variants will provide us information about the role of critical residues in the control and regulation of the PrimPol activities, which might be used to generate PrimPols variants with potential biotechnological uses.
Original text from CORDIS.
Participants
- AGENCIA ESTATAL CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS · MadridCoordinatorSpain
Links
Data: CORDIS, © European Union