Biology subjects

Blanco, L.

Publications and source records attributed to Blanco, L..

2 recordsLinked to original sources

bioRxiv2020

A unique arginine cluster in PolDIP2 enhances nucleotide binding and DNA synthesis by PrimPol

Replication forks often stall at damaged DNA. Resumption of DNA synthesis can occur by replacement of the replicative DNA polymerase with specialized, error-prone translesion DNA polymerases (TLS), that have higher tolerance for damaged substrates. Several of these polymerases (Pol{lambda}, Pol{eta} and PrimPol) are stimulated in DNA synthesis through interaction with PolDIP2, however the mechanism of this PolDIP2-dependent stimulation is still unclear. Here we show that PrimPol uses a flexible loop to interact with the C-terminal ApaG-like domain of PolDIP2, and that this contact is essential for PrimPols enhanced processivity. PolDIP2 increases PrimPols primer-template and dNTP binding affinity, which concomitantly enhances PrimPols nucleotide incorporation efficiency. This activity is dependent on a unique arginine cluster in PolDIP2 and could be essential for PrimPol to function in vivo, since the polymerase activity of PrimPol alone is very limited. This mechanism, where the affinity for dNTPs gets increased by PolDIP2 binding, could be common to all other PolDIP2-interacting TLS polymerases, i.e. Pol{lambda}, Pol{eta}, Pol{zeta} and REV1, and might be critical for their in vivo function of tolerating DNA lesions at physiological nucleotide concentrations.

biochemistry↗

bioRxiv2020

PrimPol primase mediates replication traverse of DNA interstrand crosslinks

Interstrand crosslinks (ICLs) are DNA lesions frequently induced by chemotherapy that interfere with essential processes such as replication and transcription. ICL repair may be initiated by the convergence of two replication forks at the crosslink, which results in a termination-like DNA structure recognized and processed by the Fanconi Anemia (FA) pathway. An alternative possibility to generate a suitable substrate for ICL repair involves "ICL traverse", a DNA damage tolerance mechanism in which a single fork arriving at the ICL can skip the lesion and restart DNA synthesis from a downstream point. This reaction requires FANCM translocase, the BLM/TOP3A/RMI1-2 (BTR) complex and other factors. Here we report that PrimPol, the second primase-polymerase identified in mammalian cells after Pol/Primase, interacts with BTR and participates in the ICL traverse reaction. A functional complementation assay reveals that the primase activity of PrimPol is required, confirming the need for re-priming events during ICL traverse. Genetic ablation of PRIMPOL strongly impaired this tolerance mechanism, making cells more dependent on fork convergence to initiate ICL repair. PRIMPOL KO cells and mice display hypersensitivity to ICL-inducing drugs, opening the possibility of targeting PrimPol activity to enhance the efficacy of chemotherapy based on DNA crosslinking agents.

molecular biology↗