Biology subjects

Makarova, A. V.

Publications and source records attributed to Makarova, A. V..

2 recordsLinked to original sources

bioRxiv2023

PrimPol variant V102A with altered primase and polymerase activities

PrimPol is a human DNA primase-polymerase which restarts DNA synthesis beyond DNA lesions and non-B DNA structures blocking replication. Disfunction of PrimPol in cells leads to slowing of DNA replication rates in mitochondria and nucleus, accumulation of chromosome aberrations, cell cycle delay, elevated sensitivity to DNA-damaging agents. PrimPol has been suggested to be associated with the development of ophthalmic diseases, elevated mitochondrial toxicity of antiviral drugs and increased cell resistance to chemotherapy. Here, we describe a rare missense PrimPol variant V102A with altered biochemical properties identified in patients suffering from ovarian and cervical cancer. The Val102Ala substitution dramatically reduced both the primase and DNA polymerase activities of PrimPol as well as specifically decreased its ability to incorporate ribonucleotides. We suggest that substitutions in this region would likely distort the active site and affect the catalytic activity of PrimPol.

biochemistry↗

bioRxiv2023

The role of catalytic and regulatory domains of human PrimPol in DNA binding and synthesis

Human PrimPol possesses DNA primase and DNA polymerase activities and restarts stalled replication forks protecting cells against DNA damage in nuclei and mitochondria. The zinc-binding motif (ZnFn) of the C-terminal domain (CTD) of PrimPol is required for DNA primase activity but the mechanism is not clear. In this work, we biochemically demonstrate that PrimPol initiates de novo DNA synthesis in cis-orientation, when the N-terminal catalytic domain (NTD) and the CTD of one molecule take part in catalysis. The modeling studies revealed that PrimPol uses a similar mode of initiating NTP coordination as the human primase. The ZnFn motif residue Arg417 is required for binding the 5-triphosphate group that stabilizes the PrimPol complex with a DNA template-primer. We found that PrimPol is able to efficiently initiate DNA synthesis in the absence of the link between the two domains. The ability of the NTD alone to prime DNA synthesis and a regulatory role of the RPA-binding motif in the modulation of PrimPol binding to DNA are also demonstrated.

biochemistry↗