Each year I inform the students in my Columbia University virology course that all known DNA polymerases – viral or cellular – require a primer to initiate DNA synthesis (it’s even stated in our textbook, Principles of Virology). This statement is no longer true, as shown by the discovery of two different DNA polymerases that can initiate DNA synthesis in the absence of any primer.
What is a primer? It’s a short piece of nucleic acid, either RNA or DNA, required by a polymerase to start nucleic acid synthesis. Some polymerases require a primer, others do not. Shown below is how a short primer base-pairs with a template and provides an 3’-OH for the polymerase to initiate nucleic acid synthesis:
RNA polymerase II, the enzyme that synthesizes mRNA from DNA, never requires a primer. Reverse transcriptases require a tRNA primer, while the requirement varies among RNA dependent RNA polymerases of RNA viruses.
Analysis of the genome sequence of bacteriophage NrS-1, which infects bacteria that inhabit deep sea vents revealed a gene that might encode a DNA polymerase. When synthesized in E. coli, the encoded protein displayed DNA polymerase activity in vitro. The enzyme could synthesize DNA in the absence of a primer, initiating with a dNTP. Furthermore, the enzyme recognizes a specific DNA sequence on the template to initiate de novo DNA synthesis.
Nrs-1 DNA polymerase, together with the bacteriophage helicase and single-stranded DNA-binding protein, can completely copy double- and single- stranded DNA molecules. Whether this DNA polymerase replicates the viral NRS-1 genome in infected cells has not been shown.
A second type of primer-independent DNA polymerase was identified by bioinformatic analysis of putative DNA polymerases encoded by self-replicating, mobile genetic elements. These elements are found in the genomes of diverse bacteria and in mitochondria. When synthesized in E. coli, this enzyme can synthesize long DNA efficiently in vitro in the absence of a primer.
These discoveries were made as a consequence of the explosion in the pace of genome sequencing in the past decade, which has revealed the vast genetic diversity of viruses and their hosts. Previous conclusions about the primer requirement of DNA polymerases were incorrect because they were based on studies done with a limited number of model organisms.