Mechanistic studies and engineering of reverse nucleotide polymerization

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In nature, DNA and RNA polymerases fulfill numerous essential biological functions within the cell, such as copying genetic information and transcribing it into messenger RNA (mRNA). The mRNAs are then decoded into proteins that perform many of the essential activities to sustain life. DNA and RNA copying proteins (known as polymerases) have been harnessed for technological purposes and are now widely used in biomedical research and in the biotechnology industry. Some of their applications include the sequencing of whole genomes, detection of bacterial and viral infections, paternity testing, and forensics. Molecular biology has been revolutionized since polymerases became commercially available. The patent on just one polymerase (Taq DNA polymerase) is estimated to have generated over $2 billion in royalties. All known and commercially available polymerases copy DNA or RNA exclusively in the “forward” direction. These polymerases use an existing template strand as the blueprint to create a complementary strand, which is comparable to the generation of a photograph from a photo negative image. The reverse process (i.e., capturing the photo negative from a photograph) is a necessary but highly complicated process in nature, since no known polymerase is able to copy in the "reverse" direction. Such a “reverse polymerase” would have many biomedical and biotechnological applications, such as RNA and DNA labeling in biomedical imaging, and sequencing of normally “unreadable” upstream sequences. A reverse polymerase will become an essential tool in molecular biology laboratories around the world, and will also have important clinical applications in cancer screening and diagnosis.I discovered that reverse polymerization is indeed possible. It came as a surprise that a small protein (tRNAHis guanylyltransferase, Thg1) is capable of reverse nucleotide addition. Thg1 uses the same mechanistic, structural, and catalytic features as the forward polymerases. Thg1 already contains all the components a reverse polymerase requires, including a catalytic palm domain (copying machine) and the means of positioning the nucleotide template for reverse copying. Most importantly, I discovered that certain Thg1 variants are able to catalyze the reverse copying reaction. In nature, Thg1 is limited to a specific function, yet this unique enzyme has the potential to be engineered to carry out a variety of novel activities. I will exploit the fact that Thg1 already executes reverse nucleotide addition to engineer new polymerases with applications in RNA and DNA labeling in living cells. The proposed studies will advance the field of polymerase research, as it will show how polymerization can proceed in both forward and reverse direction. The knowledge gained will lead to new enzymes for extended reverse polymerization that can be applied to overcome biotechnological challenges currently present in molecular biology and biomedical imaging.