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

Abstract

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 (Polλ, Polη 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 PrimPol's enhanced processivity. PolDIP2 increases PrimPol's primer-template and dNTP binding affinity, which concomitantly enhances PrimPol's 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 PolDIP2interacting TLS polymerases, i.e. Polλ, Polη, Polζ and REV1, and might be critical for their in vivo function of tolerating DNA lesions at physiological nucleotide concentrations.replication fork progression (4-6). Among various TLS polymerases, the primase/polymerase PrimPol has been shown to help both in nuclear and mitochondrial DNA replication fork progression (7-8). Additionally, PrimPol can also re-prime downstream of blocking lesions, thus re-reinitiating DNA replication (1,(9)(10)(11)(12)(13)(14)(15).PrimPol is a monomeric enzyme belonging to the archaeal-eukaryotic primase (AEP) superfamily (16). Its catalytic core contains three highly conserved motifs (see Figure1A) which build the dNTP binding site used for elongation (17). To start primer synthesis, PrimPol requires a unique zinc finger (ZnF)-containing C-terminal domain, which facilitates binding of the first 5´-nucleotide (1, 18). Beside its function as a primase, PrimPol behaves in vitro as a polymerase with very low processivity (9, 19), suggesting the need for a cofactor to function optimally in the cell. In contrast to some other TLS polymerases (20), PrimPol's activity is not regulated by the sliding clamp Proliferating Cell Nuclear Antigen (PCNA) (21), suggesting that another factor might control its function. Indeed, polymerase d-interacting protein 2 (PolDIP2; also known as PDIP38) and single stranded DNA binding protein Replication Protein A (RPA) (22-24) are able to stimulate PrimPol-dependent DNA synthesis. Interestingly, PolDIP2was previously shown to physically interact with TLS polymerases Polz and REV1 (25), as well as to increase the processivity of Poll and Polh (26,27). Therefore, many TLS DNA polymerases could be regulated by a common, still to be elucidated, PolDIP2dependent mechanism.Here we show that PrimPol uses a flexible loop located at its AEP catalytic core to interact with the conserved ApaG-like C-terminal region of PolDIP2 (28), which is essential for the stimul...

Crystal structure and molecular dynamics of human POLDIP2, a multifaceted adaptor protein in metabolism and genome stability

“…It should be noted however that our representation of PrimPol FL is a single predictive model, and it is feasible that the disordered PrimPol loop could stretch across to interact with the distal face of POLDIP2. This PrimPol loop also functionally interacts with Cterminal/DUF525 region (27), which is closer than the E-YccV (crosslinking) contacts in our model. This suggests that significant conformational flexibility and dynamic motion is required from both POLDIP2 and PrimPol partners, to reconcile reported functional and crosslinking features.…”

“…Sitting at the base of the domain it forms crystal contacts comprising an intermolecular antiparallel b-sheet with the equivalent b13 strand on an adjacent monomer (not shown). POLDIP2 also contains a conserved glycine-rich motif associated with pyrophosphate or nucleotide binding (GxGxxG, Figure 1) in the DUF525 region (raspberry, Figure 8a), (26), situated beside an arginine-rich cluster (27). The GxGxxG motif was not observed to bind nucleotides in DUF525 or ApaG homologues (15,16).…”

“…The GxGxxG motif was not observed to bind nucleotides in DUF525 or ApaG homologues (15,16). However, both the glycine and arginine-rich regions are important for the stimulation of PrimPol DNA synthesis, with the arginine-rich cluster also stimulating dNTP binding (27).…”

“…To gain further insight, we derived a model of POLDIP2-PrimPol interactions (Figure 8a), consistent with both the POLDIP2 crystal structure and modelling presented here, and previous reported experimental observations. We manually arranged the POLDIP2 FL and PrimPol FL protomers (based on predictive Robetta models), using known contact points from bissulfosuccinimidyl suberate (BS3) crosslinking mass spectrometry (XlMS) (6) and functional inference from biochemical analyses (27).…”

“…Following the requirement of the glycine/arginine-rich region for PrimPol stimulation described (27), the region was orientated in our model close to where the incoming dNTP binds to the primer/template (p/t) DNA in PrimPol (Figure 8a). POLDIP2 was further directionally orientated to PrimPol using XiMS contact points (6).…”