Motif WFYY of human PrimPol is crucial to stabilize the incoming 3′-nucleotide during replication fork restart

Abstract

PrimPol is the second primase in human cells, the first with the ability to start DNA chains with dNTPs. PrimPol contributes to DNA damage tolerance by restarting DNA synthesis beyond stalling lesions, acting as a TLS primase. Multiple alignment of eukaryotic PrimPols allowed us to identify a highly conserved motif, WxxY near the invariant motif A, which contains two active site metal ligands in all members of the archeo-eukaryotic primase (AEP) superfamily. In vivo and in vitro analysis of single variants of the WFYY motif of human PrimPol demonstrated that the invariant Trp87 and Tyr90 residues are essential for both primase and polymerase activities, mainly due to their crucial role in binding incoming nucleotides. Accordingly, the human variant F88L, altering the WFYY motif, displayed reduced binding of incoming nucleotides, affecting its primase/polymerase activities especially during TLS reactions on UV-damaged DNA. Conversely, the Y89D mutation initially associated with High Myopia did not affect the ability to rescue stalled replication forks in human cells. Collectively, our data suggest that the WFYY motif has a fundamental role in stabilizing the incoming 3′-nucleotide, an essential requisite for both its primase and TLS abilities during replication fork restart.

Arabidopsis thaliana PrimPol is a primase and lesion bypass DNA polymerase with the biochemical characteristics to cope with DNA damage in the nucleus, mitochondria, and chloroplast

“…In reactions harboring dGTP instead of rGTP (Fig. 2 A, lanes 5, 7, 9, and 11), AtPrimPol substantially increases the amount of synthesized dinucleotides, indicating that as human PrimPol, AtPrimPol preferentially incorporates dNTPs instead of rNTPs 21 , 37 .…”

“…bacterial DnaG and eukaryotic PriS/Prim1, respectively) synthesize short RNA primers for their cognate DNAPs 36 . In contrast PrimPol has a remarkable ability to synthesis DNA primers using dNTPs instead or NTPs 21 , 37 . Because of the traditional role of primases in synthetizing RNA primers, we decided to investigate if AtPrimPol was able to use solely NTPs as a substrate.…”

3′dNTP Binding Is Modulated during Primer Synthesis and Translesion by Human PrimPol

“…In addition to these direct ligands, there are other residues that indirectly contribute to the stabilization of the 3′ incoming dNTP by Hs PrimPol. For example, the interaction network maintained by both Trp 87 and Tyr 90 induces a torsion of β-strand 11, so that Arg 291 and other amino acids such as Lys 297 and Lys 300 are able to contact the 3′ incoming dNTP [ 26 ].…”