Human PrimPol Discrimination against Dideoxynucleotides during Primer Synthesis

• Published in: Genes 2021-09, Vol.12 (10), p.1487
• Main Authors: Carvalho, Gustavo, Díaz-Talavera, Alberto, Calvo, Patricia A, Blanco, Luis, Martínez-Jiménez, María I
• Format: Article
• Language: English
• Subjects: Acquired immune deficiency syndrome; AIDS; Amino Acid Sequence - genetics; Crystal structure; Dideoxynucleotides - genetics; DNA biosynthesis; DNA polymerase; DNA Primase - genetics; DNA Primers - chemical synthesis; DNA Primers - genetics; DNA Replication - genetics; DNA, Mitochondrial - genetics; DNA-Directed DNA Polymerase - genetics; Elongation; Humans; Kinases; Manganese; Mitochondria; Mitochondrial DNA; Multifunctional Enzymes - genetics; Mutation; Nucleotides - genetics; Primase; Primers; Replication; Toxicity
• ISSN: 2073-4425; 2073-4425
• DOI: 10.3390/genes12101487

PrimPol is required to re-prime DNA replication at both nucleus and mitochondria, thus facilitating fork progression during replicative stress. ddC is a chain-terminating nucleotide that has been widely used to block mitochondrial DNA replication because it is efficiently incorporated by the replicative polymerase Polγ. Here, we show that human PrimPol discriminates against dideoxynucleotides (ddNTP) when elongating a primer across 8oxoG lesions in the template, but also when starting synthesis of DNA primers, and especially when selecting the 3'nucleotide of the initial dimer. PrimPol incorporates ddNTPs with a very low efficiency compared to dNTPs even in the presence of activating manganese ions, and only a 40-fold excess of ddNTP would significantly disturb PrimPol primase activity. This discrimination against ddNTPs prevents premature termination of the primers, warranting their use for elongation. The crystal structure of human PrimPol highlights Arg residue as responsible for the strong dNTP/ddNTP selectivity, since it interacts with the 3'-OH group of the incoming deoxynucleotide, absent in ddNTPs. Arg , shown here to be critical for both primase and polymerase activities of human PrimPol, would contribute to the preferred binding of dNTPs ddNTPs at the 3'elongation site, thus avoiding synthesis of abortive primers.