Abstract
Replication forks often stall at damaged DNA. To overcome these obstructions and complete the DNA duplication in a timely fashion, replication can be restarted downstream of the DNA lesion. In mammalian cells, this repriming of replication can be achieved through the activities of primase and polymerase PrimPol. PrimPol is stimulated in DNA synthesis through interaction with PolDIP2, however the exact 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 PrimPol's enhanced processivity. PolDIP2 increases primer-template and dNTP binding affinities of PrimPol, which concomitantly enhances its nucleotide incorporation efficiency. This stimulation is dependent on a unique arginine cluster in PolDIP2. Since the polymerase activity of PrimPol alone is very limited, this mechanism, where the affinity for dNTPs gets increased by PolDIP2 binding, might be critical for the in vivo function of PrimPol in tolerating DNA lesions at physiological nucleotide concentrations.
Highlights
In all organisms, the bulk of genomic DNA replication is done by replicative DNA polymerases
An earlier study showed that human PolDIP2 increases the DNA polymerase activity of PrimPol [22]
Addition of 300–500 nM T1-PolDIP2 showed a modest increase in PrimPol DNA synthesis (Figure 1C, compare lane 2 with lanes 8 and 9; Figure 1D), but this T1 stimulation was substantially weaker compared to the effect of T3- or FL-PolDIP2 (Figure 1C, compare lanes 7–9 with lane 15–17; Figure 1D)
Summary
The bulk of genomic DNA replication is done by replicative DNA polymerases These enzymes, characterized by their high processivity and fidelity, occasionally encounter obstacles on the DNA that significantly delay their progression, such as damaged DNA. If not alleviated, this can lead to fork collapse and genomic instability. In order to prevent genomic instability, cells have developed several DNA damage tolerance mechanisms that help the replication fork dealing with various disturbances and allow completion of the replication process. Polymerase ␦-interacting protein 2 (PolDIP2; known as PDIP38) and single stranded DNA binding protein Replication Protein A (RPA) [22,23,24] are able to stimulate PrimPol-dependent DNA synthesis. The PolDIP2-dependent mechanism of PrimPol polymerization stimulation has not yet been fully clarified
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