This part (terminal protein DNAP1) represents a DNA polymerase which was extracted from the K. lactis – a yeast strain actively used in biotechnology (especially fermentation {especially cheese production}) and science Bergquist2002. This strain is also known to harbor and express a linear cytoplasmic yeast plasmids. This part is an element of the Orthorep system, where it is responsible for replicating the engineered plasmid with the genes of interest. We used a yeast strain harbouring the aforementioned polymerase and linear plasmid, which was developed for increased error rate of the polymerase and characterized by the group of Chang Liu Zhong2018, increasing its mutation rate. Since the common yeast strains employed in research (S. cerevisiae) are lacking linear cytoplasmic plasmids in normal conditions, the insertion of this polymerase, together with the engineered OrthoRep system allows to orthogonally mutagenize genes of interest in vivo Ravikumar2018.

To better understand the system, we first sought to take a look at the structure of our polymerase. As for general characteristics, the polymerase consists out of 995 amino acids and has a mass of around 116 kDa. A search for more structure-related information in the literature turned up blank, and even finding our protein in Uniprot left us without a structure. Thus, we had to take matters into our own hands and attempt to solve the structure of TP-DNAP1. We proceeded to build homology models of the protein using SwissModel.

There, we were met with a pitiful amount of homology to our protein, which reliably drove model quality into the ground. Another difficulty arose due to the lack of homological structures for a large part of the C-terminus of our protein (first 325 AA were thus not included in the model, that correspond to the terminal protein sequence). Now, we had two options left: solve our structure de novo or crystallize, and we sure as heck wouldn't go for the latest option. Expectedly we moved on to greener pastures and submitted our sequence to both Robetta and RaptorX. The results of the de-novo structure assembly can be seen in the images below.