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Artificial evolution of a novel multifunctional human polymerase

Periodic Reporting for period 1 - EVOPRIMPOL (Artificial evolution of a novel multifunctional human polymerase)

Reporting period: 2015-05-01 to 2017-04-30

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.
Selection or residues to be randomized was based on previous biochemical data obtained in our laboratory. Recently, we have observed the critical role of a structural motif in HsPP (residues from Trip87 to Tyr90; WFYY motif) for both priming and polymerization reactions due to its contribution to the stabilization of the incoming nucleotide. We decided to randomize residues F88 and Y89 in order to boost the activity of this enzyme and characterize how this motif modulates HsPP activities.

Double-site library in positions F88 and Y89 (“88-89”) has been constructed using NBT (N: adenine, cytosine, guanine, thymine; B: cytosine, guanine, thymine T: thymine) and NDT (D: adenine, guanine, thymine;) degeneracy, respectively. These randomizations encodes for 11 (NBT) and 12 (NDT) different amino acidos, that rendered a library composed by 132 different variants.
The library has been prepared by mutagenic PCR. This reaction has been carried out using commercially prepared primers that contain the corresponding degenerated sequence in the codon that encodes the target amino acid. As template, the plasmid encoding the wild-type WT HsPP sequence has been used. After PCR reaction, codon variability generated in the library was assessed by a quality test.

For mutant selection, we have envisioned a new screening method based on an enhanced fluorescence emission by the chromophore SYBR GreenI (SGI) upon interaction with double strand DNA (dsDNA). Thus, At the start of the assay, SGI emits a low fluorescence background due to the presence of the unreplicated single strand DNA (ssDNA) template; however, as the reaction proceeds HsPP primase-polymerase activity will produce dsDNA, optimal for dye intercalation, thus increasing brightness of SGI, that could be recorded in real time kinetics using a fluorimeter. This method was successfully validated using both purified HsPP (Fig. 1) and crude E. coli extracts overexpressing different recombinant HsPP variants in a 96-well plate platform (Fig. 2). An advantage of this screening method is the specificity of the HsPP DNA priming reaction, since there is not other protein in E. coli displaying DNA primase activity on a DNA template.

We then subjected the 88-89 library to a modified screening protocol: instead of using a ssDNA as template, we have used a template that contains the RNA version of the HsPP preferential priming site. Thus, library 88-89 was tested for an enhanced DNA primase-polymerase on an RNA template (RdDP).

In the initial fluorometric screening, 18 HsPP variants showing increased RdDP activities were shortlisted. Selected variants were overexpressed in E. coli BL21(DE3)-pRIL cells, purified and further evaluated to confirm the RdDP improvement. Biochemical primase experiments confirmed an enhanced RdDP activity for 9 variants. The one showing the largest increase in RdDP was Y89R mutant (Fig. 3).
This mutant displays at least 10-fold higher RdDP activity than WT HsPP. Y89R variant has been biochemical characterized using enzymological assays previously validated in the laboratory of Prof. Blanco for characterization of HsPP. The results obtained in these assays indicated that the enhanced RdDP activity in mutant Y89R is due to an increased stabilization of the preternary complex (HsPP:dNTP:DNA) that precedes dimer formation (i.e. priming event) whereas this mutation only has a minor effect on template binding and nucleotide incorporation during the further polymerization.
a) We have developed a screening platform to assess different HsPP variants that specifically discriminates DNA-priming-DNA polymerase activity from other related activities displayed by regular primases. The high specificity and selectivity of the assay to detect HsPP primase has a high potential to evolve and enhance HsPP recognition and use of natural and chemically-modified nucleotides as substrates. Furthermore, slight modifications of this method might open the use of the platform to check HsPP activity in the presence of inhibitors and drugs, and is likely extrapolable to other primases and polymerases. These aforementioned assays may be relevant to develop biotechnological tools, anticancer therapies and high-throughput analysis of polymerases mutant libraries (Agudo et al., submitted 2nd revision).

b) This study has identified Y89R as a mutation that improves the stabilization of the complex HsPP:dNTP:DNA, as an intermediate preceding primer synthesis The improved activity of mutant Y89R on RNA templates allowed its use to prime RT reactions, when combined with a reverse transcriptase. Thus, this study represents a proof of principle of the potential biotechnological use of HsPrimPol as a DNA primase, as well as exploits the power of direct evolution not just to improve or alter its enzymatic properties, but to unveil its intimate structure-function details.

c) Thanks to our promising results, Prof. Blanco lab has obtained a grant from the Spanish Ministerio de Economia y Competitividad to develop, in collaboration with the biotechnological company Expedeon, a platform for the screening of mutants from the Thermus thermophilus PrimPol (TthPP) based on the method developed in EVOPRIMPOL. TthPP is an enzyme that is already successfully commercialized by Expredeon as a part of the revolutionary amplification method system TruePrime. The objective of this collaboration is the generation of PrimPol variants with altered biochemical properties for their biotechnological use.