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.
