An integrative biology approach to study DNA primase: the swiss-knife of Archaea – ARCHAPRIM

The DNA of all organisms is constantly damaged by exogeneous and endogenous agents. A complex interplay between DNA replication, recombination and repair is required to ensure faithful passage of the genetic material from one generation to the next. Our understanding of how these DNA replication, recombination and repair components work and interact together at the molecular level is still fragmentary and constitute one of the wonders of molecular biology. Hyperthermophilic Archaea constitute an attractive model for studying the interplay between DNA replication and repair.
In Euryarchaea, the DNA primase-polymerase forms a heterodimeric complex (PriSL) containing both a small catalytic subunit (PriS) and a larger accessory subunit (PriL), both subunits being essential for cell viability. The DNA primase plays an essential role in replication initiation by generating RNA primers that enable DNA synthesis to be initiated by DNA polymerases, which are otherwise unable to initiate DNA synthesis de novo. Recently, a growing number of evidences have shown that activities and cellular roles of the archaeal primase extends further than this essential role in initiating DNA replication. The archaeal primase is capable of accomplishing an astonishing range of polymerisation activities, being able of DNA and RNA priming activities, DNA-dependent DNA and RNA polymerization, strand-displacement and lesion bypass DNA synthesis. The archaeal primase belongs to the archaeo-eukaryotic primase (AEP), which has been recently reclassified under a category called primase-polymerases to better recognize their wide variety of cellular roles including replication, repair and damage tolerance, in addition to primer synthesis.
Many questions remain about the molecular mechanism and regulation of PriSL’s intertwined DNA replication and DNA repair activities. How does the PriSL active site cope with such an astonishing range of polymerisation activities ? What is the role of PriL in regulating the primase activity? Can the 4Fe-4S cluster located in the C-terminal region of PriL act as a molecular sensor, which regulates the DNA replication and DNA repair activities of PriSL upon oxidative stress. What are the molecular basis for the cooperativity between PriSL and the replisome machinery, and how do these interactions regulate PriSL activity by addressing it toward DNA replication or DNA repair ? This proposal aims to answer these questions by using a multi-disciplinary approach, which combines structural biology studies, primarily by X-ray crystallography and cryo-electron microscopy, and a wide range of in vitro activity assays along with in vivo genetic analyses.
This proposal aims to approach fundamental questions in DNA replication and repair by using hyperthermophilic Archaea as a model system. The expected results of this proposal will shed light on the origin of the replication system in the tree of life, including eukaryotes, and should be of great interest for a large community of molecular biologists; in addition it will help devise new biotechnological applications of these thermophilic enzymes. Indeed, PriSL is a swiss-knife, capable of accomplishing an astonishing range of DNA and RNA polymerisation activities. Its ability of copying DNA without the need for a primer, in combination with thermostability, may be useful for DNA amplification. The ongoing COVID-19 pandemic recently demonstrated how important it is to study thermostable polymerases used in polymerase chain reaction (PCR), which is the most common way to diagnose coronavirus.