The role of human PrimPol active site residue Gln48 in catalysis and complex formation with DNA

Abstract

Human PrimPol is a primase-polymerase involved in the mechanism of DNA synthesis reinitiation at the sites of DNA damage and replication fork collapse. By using its DNA primase activity, PrimPol synthesizes DNA primers which can be utilized by high-fidelity DNA polymerases to resume replication downstream of the damaged site. Disruption of PrimPol function may be associated with an increased risk of cancer and ophthalmologic diseases. Understanding the detailed mechanism of catalysis and regulation of PrimPol activity is crucial for predicting how mutations and polymorphisms affect enzyme function within the cell. In this study, we conducted a biochemical investigation of the role of the conserved Gln48 residue in the PrimPol active site in DNA synthesis by replacing glutamine with a positively charged arginine (Q48R) or a negatively charged glutamic acid (Q48E). The PrimPol variant with the Q48R substitution is also represented in the NCBI dbSNP (rs939272279 A/G). Both substitutions resulted in impaired complex formation of PrimPol with DNA and significantly reduced the catalytic activity of the enzyme, indicating the important role of the Gln48 residue in forming contacts with the template DNA and active site organization. The data obtained suggest that the Q48R substitution may disrupt PrimPol functions.