Sunday, November 30, 2008

A challenge for the biochemist- the priming problem

All cellular life forms and many DNA viruses, phages and plasmids use a primase to synthesize a short RNA primer with a free 3' OH group that is subsequently elongated by a DNA polymerase. The existence of this process is one of the most fascinating unsolved problems. Essentially, it is unclear why DNA polymerases require a primer to initiate DNA replication, while RNA polymerases do not. From an evolutionary perspective, although DNA polymerases have evolved on multiple occasions independently, and a variety of independent solutions have evolved to address the priming problem, in no case is the DNA polymerase rid of a primer. Could this reflect something more fundamental?

First, let us review the known solutions to the priming problem.
  1. In bacteria and archaeo-eukaryotes (a term clubbing archaea and eukaryotes) that possess DNA polymerases of unrelated folds, two unrelated families of primases synthesize RNA primers. Bacteria possess a DNAG-like primase of the Toprim fold, whereas in a comprehensive sequence-structure analysis, we showed that the archaeo-eukaryotic primase (AEP) belongs to the RRM fold (click here to read).
  2. In certain plasmids and phages/viruses, a primpol protein performs both the RNA polymerase (for primer synthesis) and DNA polymerase (for DNA synthesis) activities. Primpols belong to two distinct families, one that is experimentally confirmed and belongs to the archaeo-eukaryotic primase superfamily, and the second, which awaits experimental verification, belongs to the TV-Pol family.
  3. Retroelements (including retroviruses and other reverse-transcriptase based DNA mobile elements) prime DNA replication by using a tRNA that provides a free 3' OH that is used for elongation by the reverse transcriptase (RNA-dependent DNA polymerase).
  4. In adenoviruses and the φ29 family of bacteriophages,a hydroxyl group is provided by the side-chain of an amino acid of the genome attached terminal protein to which nucleotides are added by the DNA polymerase to form a new strand.
  5. Another solution to the priming problem is seen in several families of DNA viruses, such as parvoviruses, geminiviruses and circoviruses, and many phages and plasmids. All of these replicate their DNA by rolling circle replication (RCR). Here, the RCR endonuclease (RCRE) creates a nick in one of the DNA strands. The 5' end of the nicked strand is transferred to a tyrosine residue on the nuclease, and the free 3' OH group is elongated by a DNA polymerase for the new strand synthesis.
  6. The only known exception of a DNA polymerase lacking a primer, is the reverse transcriptase of the Mauriceville plasmid that uses the 3' tRNA-like structure of the parent mRNA/pRNA to de novo synthesize a daughter DNA strand.
Thus although DNA polymerases have evolved on multiple occasions independently, they don't seem to have rid themselves of the need for a primer.

Hypothesis. In our study on the evolutionary history of the archaeo-eukaryotic primases, we speculated that these observations suggests a strong constraint against ‘invention’ of de novo initiation of DNA synthesis which, probably, stems from fundamental chemical differences between ribo- and deoxyribonucleotides, rather than a frozen evolutionary accident that maintains a primer. We proposed that the inefficiency in de novo DNA synthesis by DNA polymerases may, at least in part, be due to a competing futile reaction of 3'->5' nucleotide cyclization while using deoxyribonucleotides. Given the tendency of diverse, unrelated RNA polymerases to initiate de novo strand synthesis, it seems likely that this problem does not arise with ribonucleotides. As support for this hypothesis we note that the DNA cyclases are specifically related to DNA polymerases and have evolved from the latter on multiple occasions independently.

To the best of our knowledge this hypothesis has not yet been tested. You can read about our comprehensive study on the archaeo-eukaryotic primases by clicking here.