Primase-polymerases are a functionally diverse superfamily of replication and repair enzymes

Abstract

Until relatively recently, DNA primases were viewed simply as a class of proteins that synthesize short RNA primers requisite for the initiation of DNA replication. However, recent studies have shown that this perception of the limited activities associated with these diverse enzymes can no longer be justified. Numerous examples can now be cited demonstrating how the term 'DNA primase' only describes a very narrow subset of these nucleotidyltransferases, with the vast majority fulfilling multifunctional roles from DNA replication to damage tolerance and repair. This article focuses on the archaeo-eukaryotic primase (AEP) superfamily, drawing on recently characterized examples from all domains of life to highlight the functionally diverse pathways in which these enzymes are employed. The broad origins, functionalities and enzymatic capabilities of AEPs emphasizes their previous functional misannotation and supports the necessity for a reclassification of these enzymes under a category called primase-polymerases within the wider functional grouping of polymerases. Importantly, the repositioning of AEPs in this way better recognizes their broader roles in DNA metabolism and encourages the discovery of additional functions for these enzymes, aside from those highlighted here.

Figure 1.

Architecture of AEP catalytic subunits from the major domains of life. Representative examples of the crystal structures of AEPs that have been elucidated. Human primase subunit (Prim1) (PDBID: 4RR2), sky blue. The primase small catalytic subunit (PriS/Prim1) from the archaeal species Pyrococcus horikoshi (PDBID: 1V33), pale crimson. The NHEJ repair polymerase (PolDom/LigD-Pol) from Mycobacterium tuberculosis (PDBID: 2IRU), magenta. The AEP domain of RepB’ encoded by the Escherichia coli plasmid RSF1010 (PDBID: 3H20), gold. The AEP domain of ORF904 encoded by the Sulfolobus islandicus plasmid pRN1 (PDBID: 3M1M), sea green. The conserved catalytic core of these enzymes is shown in a lighter hue and catalytic triads are rendered as sticks with the acidic oxygens coloured red. Where present, the coordinated zinc atoms in the zinc finger domains are coloured tan.

Figure 2.

Domain organization of members of the AEP superfamily. The AEP superfamily is formed of a number of divergent enzymes with varying domain organizations. Some representative examples of members of the AEP superfamily are displayed here with the three signature catalytic motifs of the AEPs depicted in red, in addition to any accessory domains associated with this domain. The blue, red, green and purple backgrounds correspond to the different domains of life to which these primase family members belong.

Figure 3.

Alternative hypotheses for the evolution of AEP and Toprim primases. (A) The first model of primase evolution suggests that primases evolved independently twice from a last universal common ancestor (LUCA). Bacterial ancestors evolved toprim primases and archaeal and eukaryotic ancestors evolved AEP primases. Subsequent horizontal gene transfer occurred between the two lineages to account for AEP primase's role in NHEJ in bacteria and toprim-type primase's role in archaeal RNA degradation. (B) The second model of primase evolution suggests that LUCA had a dual primase replication mechanism, consisting of both AEP and toprim primases. During the evolution of bacteria, they lost the replicative function of the AEP primases but retained them for the auxiliary function of NHEJ-mediated DNA repair. During the evolution of the archaeal/eukaryotic lineage, the replicative function of toprim primases was lost but their auxiliary role in archaeal RNA degradation was retained. (C) The third model of primase evolution suggests that LUCA had either an AEP or toprim-like primase. Significant, evolutionary pressures could then have driven the evolution or acquisition of a second class of primase. (D) 12 of the 13 major AEP families can be arranged into three higher order clades, the AEP Proper Clade, the NCLDV-Herpesvirus primase clade and the Prim-Pol clade.

Figure 4.

Nucleotidyltransferase activities associated with AEP members. AEP-type primase family members possess many more activities, in addition to catalyzing primer synthesis DNA for replication. The reported additional nucleotidyltransferase activities for each of the different AEPs are depicted, including polymerase activity (either DNA-dependent DNA polymerase or DNA dependent RNA polymerase), lesion bypass, terminal transferase and strand-displacement. The observed ability of each enzyme to perform the indicated activity is noted by a tick. The blue, red, green and purple backgrounds correspond to the domain of life in which the primase family is found.

Figure 5.

Structures of AEPs bound to DNA substrates. Structural examples of AEP members bound to DNA intermediates. (A) Structure a pre-ternary catalytic conformation of a NHEJ repair polymerase (PolDom/LigD-Pol) from Mycobacterium tuberculosis bound to a ds DNA break with a 3′ overhanging terminus (PDBID: 4PKY, ice blue) with UTP and manganese cofactors, coloured cyan and pink, respectively. (B) Crystal structure of a micro-homology mediated end-joining (MMEJ) intermediate showing an NHEJ repair polymerase-mediated synapsis of a DSB (PDBID: 4MKY, ice blue and lemon). (C) Structure of the AEP domain of RepB’ bound to a ssiA DNA replication initiation site (PDBID: 3H25, lawn green). The catalytic residues are rendered as sticks with the acidic oxygens coloured red. DNA strands are coloured red or green.

Figure 6.

Diversity of functional roles fulfilled by AEPs. AEP superfamily members are employed in many different biological roles, in addition to replicative primases. These enzymes are also utilized as primer extenders, plasmid replicases, damage-tolerance re-priming enzymes, TLS polymerases, NHEJ DNA break repair and terminal transferase polymerases.