The Zn-finger domain of human PrimPol is required to stabilize the initiating nucleotide during DNA priming

Abstract

Human PrimPol is a monomeric enzyme whose DNA primase activity is required to rescue stalled replication forks during nuclear and mitochondrial DNA replication. PrimPol contains an Archeal-Eukaryotic Primases (AEP) core followed by a C-terminal Zn finger-containing domain (ZnFD), that is exclusively required for primer formation and for PrimPol function in vivo. The present study describes the sequential substrate interactions of human PrimPol during primer synthesis, and the relevance of the ZnFD at each individual step. Both the formation of a PrimPol:ssDNA binary complex and the upcoming interaction with the 3′-nucleotide (pre-ternary complex) remained intact when lacking the ZnFD. Conversely, the ZnFD was required for the subsequent binding and selection of the 5′-nucleotide that will become the first nucleotide of the new primer strand. Providing different 5′-site nucleotides, we can conclude that the ZnFD of PrimPol most likely interacts with the γ-phosphate moiety of the 5′-site nucleotide, optimizing formation of the initial dimer. Moreover, the ZnFD also contributes to recognize the cryptic G at the preferred priming sequence 3′GTC5′. Dimer elongation to obtain long DNA primers occurs processively and is facilitated by the 5′-terminal triphosphate, indicating that the ZnFD is also essential in the subsequent translocation/elongation events during DNA primer synthesis.

Identifying the role of PrimPol in TDF-induced toxicity and implications of its loss of function mutation in an HIV+ patient

“…Although D114N PrimPol cannot form the initial dinucleotide for primer synthesis, it may retain the ability to elongate preexisting primers. To test that, we supplied the reaction with a synthetic 3-mer primer with a 5′-triphosphate, (3pAGT), which has previously been demonstrated to be important for the binding of PrimPol to the initiated primer 39 . In these conditions, extension of the primer was efficiently carried out by wild type PrimPol but not by the D114N mutant ( Fig.…”

PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts

“…PrimPol exerts both DNA/RNA primase and DNA polymerase activity 10 . To test whether the PrimPol primase activity is specifically required for continued replication fork progression in presence of BPDE, we performed rescue experiments by ectopically expressing wild type (WT) and primase-dead (CH) mutant versions of PrimPol 11,32 (Fig. 2h).…”

Divalent Cations Alter the Rate-Limiting Step of PrimPol-Catalyzed DNA Elongation

“…Considering that manganese is known to accumulate in brain and liver mitochondria [35,36] and that PrimPol potentially plays a role in mitochondrial DNA replication [13,20], it is conceivable that PrimPol's enzymatic activity can be stimulated by Mn 2+ under certain conditions. A recent report focused on PrimPol's primase activity also supports in the importance of Mn 2+ in forming pre-ternary complexes in PrimPol-mediated DNA priming [24].…”

“…PrimPol is a 560-amino acid protein that has an N-terminal AEP-like catalytic domain and a C-terminal zinc finger domain. The catalytic domain is responsible for the nucleotidyl transferase activity of PrimPol, and the zinc finger domain facilitates the binding and selection of the 5′-nucleotide of the newly synthesized primer and the recognition of preferred initiation sites [24]. The C-terminal domain also includes crucial motifs that interact with replication protein A (RPA) [25].…”