Human PrimPol activity is enhanced by RPA

Abstract

Human PrimPol is a primase belonging to the AEP superfamily with the unique ability to synthesize DNA primers de novo, and a non-processive DNA polymerase able to bypass certain DNA lesions. PrimPol facilitates both mitochondrial and nuclear replication fork progression either acting as a conventional TLS polymerase, or repriming downstream of blocking lesions. In vivo assays have shown that PrimPol is rapidly recruited to sites of DNA damage by interaction with the human replication protein A (RPA). In agreement with previous findings, we show here that the higher affinity of RPA for ssDNA inhibits PrimPol activities in short ssDNA templates. In contrast, once the amount of ssDNA increases up to a length in which both proteins can simultaneously bind ssDNA, as expected during replicative stress conditions, PrimPol and RPA functionally interact, and their binding capacities are mutually enhanced. When using M13 ssDNA as template, RPA stimulated both the primase and polymerase activities of PrimPol, either alone or in synergy with Polε. These new findings supports the existence of a functional PrimPol/RPA association that allows repriming at the exposed ssDNA regions formed in the leading strand upon replicase stalling.

Figure 1

Modulation of PrimPol polymerase activity by RPA. (a) Effect of RPA on PrimPol polymerase activity on a short template/primer structure (30-mer/16-mer), either with an undamaged control template (left panel) or with a template containing a thymine dimer 6-4 photoproduct (right panel). PrimPol (200 nM) was incubated either alone or with RPA (12, 50 and 200 nM) in buffer R supplemented with 100 µM dNTPs and 2 nM labeled DNA. (b) Effect of RPA on PrimPol polymerase activity on a longer template/primer structure (65-mer/16-mer). PrimPol (200 nM) was incubated either alone or with RPA (12, 50 and 200 nM) in buffer R supplemented with 100 µM dNTPs and 2 nM labeled DNA. (c) Effect of RPA on PrimPol polymerase activity on circular M13 ssDNA annealed to a labeled oligonucleotide. PrimPol (200 nM) and RPA (20, 100, 500 nM) when indicated, were incubated with dNTPs (100 µM), 5 mM MgCl₂ and 1 mM MnCl₂ as metal cofactors, and primed-M13 ssDNA (2.5 nM). When indicated, PrimPol and RPA were pre-incubated 10 min at RT before template/primer addition. This experiment has been repeated more than 3 times, showing the same effect in all cases. The scheme represents the stimulation of PrimPol polymerase activity when increasing concentrations of RPA are added to the reaction when ssDNA is covered by RPA, PrimPol molecules cannot be unproductively bound, but form a recycling pool to fuel binding and extension of the primer (1); besides, a specific PrimPol/RPA association (2) can directly enhance the rate and processivity of polymerization.

Figure 2

Modulation of PrimPol primase activity by RPA. The inhibitory effect of RPA on PrimPol primase activity was evaluated using two preferred templates differing in their size: (a) 29-mer GTCC or (b) 60-mer GTCC. The primase reaction contained 16 nM [γ-³²P]ATP, dGTP (1, 10, 100 µM), 5 mM MgCl₂ and 1 mM MnCl₂ as metal cofactors, 1 µM of the indicated template, PrimPol (200 nM), and RPA (12, 50, 200 nM) when indicated. (c) RPA stimulates PrimPol primase activity on a heterogeneous 65-mer oligonucleotide. The reaction contained t16 nM [α-³²P]dGTP, 100 µM of dATP, dCTP and dTTP as indicated, 5 mM MgCl₂ and 1 mM MnCl₂ as metal cofactors, PrimPol (200 nM), and RPA (200 nM) when indicated. The schemes depict the template oligonucleotide, PrimPol (grey square), and the synthesized primer product.

Figure 3

Mutual interaction of PrimPol and RPA with ssDNA. EMSA carried out as described in Materials and Methods, in the presence of a 65-mer ssDNA (2 nM) and the indicated concentration of PrimPol (a), RPA (b), or a combination of both proteins (c). The different retarded bands, representing individual protein:ssDNA complexes, were resolved by electrophoresis and autoradiography.

Figure 4

RPA stimulates both primase and polymerase activities of PrimPol in synergy with Polε. (a) Effect of RPA on the combined primase and polymerase activities of PrimPol in non-primed M13 ssDNA. PrimPol (200 nM) was incubated with the template M13 ssDNA 2 nM, 100 µM of dATP, dCTP, dTTP and GTP, 16 nM [α-³²P] dGTP in the presence of 5 mM MgCl₂ and 100 µM MnCl₂ as metal cofactors; when indicated, RPA was added to the reaction at a concentration of 100 or 500 nM. (b) PrimPol-Polε combined assay was carried out essentially as described in part (a) but using 50 µM MnCl₂, and when indicated adding Polε (10 nM) and RPA (20, 100 or 500 nM). After native agarose electrophoresis, the nature of the labelled products (circular ssDNA and linear ssDNA) was inferred from EtBr-staining of the gels prior to autoradiography. The schemes represent the stimulation of PrimPol primase and polymerase activities in the presence of RPA, and the subsequent stimulation on extension by Polε.

Figure 5

Model for PrimPol and RPA interactions at the replication fork during replicative stress. During normal replication fork progression, the leading replicase and the helicase are coordinated, RPA mainly binds to the lagging strand and PrimPol (depicted in association to RPA) has no access to the short ssDNA ahead of the leading replicase (likely covered by RPA). Under replicative stress, the leading strand replicase is stalled, and PrimPol/RPA now can gain access to the long stretches of ssDNA accumulated as a consequence of a sustained helicase unwinding, in a way compatible with an improved binding of RPA to ssDNA. PrimPol repriming in the leading strand triggers a polymerase switch that mobilizes the leading strand replicase from the stalling site to the new DNA primer, that becomes elongated, and the excess of RPA becoming displaced and dissociated. Recovery of the coordination between replicase and helicase re-establishes fork progression and normal lagging strand synthesis. The gap left behind in the leading strand constitutes a damage tolerance scenario, now accessible to translesion and/or repair machineries. For simplicity, the primase and polymerases acting on the lagging strand are not represented. RNA primers in the lagging strand (green) or an eventual DNA primer made by PrimPol in the leading strand (cyan) are numbered according to their proposed order of synthesis.