Structure and mechanism of human PrimPol, a DNA polymerase with primase activity

Rechkoblit, Olga; Gupta, Y. K.; Malik, Radhika; Rajashankar, Kanagalaghatta R.; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

doi:10.1126/sciadv.1601317

Cited by 70 publications

(123 citation statements)

References 44 publications

(99 reference statements)

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“…This result is consistent with previous work and confirms that FL-PolDIP2 is able to stimulate PrimPol during DNA synthesis (22). Primer-extension reactions in the presence of the PolDIP2 fragments showed the PrimPol stimulation is mainly dependent on fragment T3 ( Figure 1C, lanes [15][16][17] and to a lesser extent on T1 ( Figure 1C, lanes 7-9), but not on T2 ( Figure 1C, lanes [11][12][13]. Even the lowest concentration of T3-PolDIP2 fragment (150 nM; Figure 1C, lane 15) showed a robust stimulation of DNA synthesis.…”

Section: The C-terminal Region Of Poldip2 Increases the Processivity supporting

confidence: 92%

“…To investigate if the presence of PolDIP2 influences dNTP binding by PrimPol, we performed filter binding assays in the presence of a DNA primer/template and the complementary nucleotide for the first position after the primer, [a-32 P]dCTP. Ca 2+ was used as the metal cofactor, since it supports PrimPol binding to the incoming dNTP, but not primer extension ( Figure 5A) (17,40). As expected, the dNTP binding capacity of PrimPol alone was much lower when compared with Pol g, the replicative mitochondrial DNA polymerase (compare the y-axis scale of Figure 5A and 5B), and PolDIP2 alone was very inefficient in binding dCTP ( Figure 5A).…”

Section: The Arginine Cluster In Poldip2 Favors Dntp Binding By Primpolmentioning

confidence: 80%

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

Kasho

Stojkovič

Velázquez-Ruiz

et al. 2020

Preprint

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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...

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Section: The C-terminal Region Of Poldip2 Increases the Processivity supporting

confidence: 92%

Section: The Arginine Cluster In Poldip2 Favors Dntp Binding By Primpolmentioning

confidence: 80%

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

Kasho

Stojkovič

Velázquez-Ruiz

et al. 2020

Preprint

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“…1a). The exceptions are Y-family polymerases, which have an additional Little-Finger domain (LF, also known as PAD) essential for DNA binding (12, 50), and PrimPol members, which lack a distinct thumb domain (51). The catalytic center of all DNA polymerases resides in the palm domain and usually contains three carboxylates.…”

Section: Dna Polymerases For Replication and Repairmentioning

confidence: 99%

Translesion and Repair DNA Polymerases: Diverse Structure and Mechanism

Yang

Gao

2018

Annu. Rev. Biochem.

182

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The number of DNA polymerases identified in each organism has mushroomed in last two decades. Most newly found DNA polymerases specialize in translesion synthesis and DNA repair instead of replication. Although intrinsic error rates are higher for translesion and repair polymerases than for replicative polymerases, the specialized polymerases increase genome stability and reduce tumorigenesis. Reflecting the numerous types of DNA lesions and variations of broken DNA ends, translesion and repair polymerases differ in structure, mechanism and function. Here we review the unique and general features of polymerases specialized in lesion-bypass, gap-filling and end-joining synthesis.

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“…Unlike most primases that are heterodimeric and utilize NTPs, PrimPols are monomers that prefer dNTPs for de novo nucleic acid synthesis. Furthermore, PrimPols can extend primers, even across UV-induced lesions, and both primase and DNA polymerase activities are catalyzed by the same active site 73, 74, 140–142 . In humans, the highly-conserved C-terminus of PrimPol is required for its interaction with RPA 139, 143 .…”

Section: The Gps For Tls: Correlation Between Re-priming the Damagmentioning

confidence: 99%

Eukaryotic Translesion DNA Synthesis on the Leading and Lagging Strands: Unique Detours around the Same Obstacle

Hedglin

Benkovic

2017

Chem. Rev.

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During S-phase, minor DNA damage may be overcome by DNA damage tolerance (DDT) pathways that bypass such obstacles, postponing repair of the offending damage to complete the cell cycle and maintain cell survival. In translesion DNA synthesis (TLS), specialized DNA polymerases replicate the damaged DNA, allowing stringent DNA synthesis by a replicative polymerase to resume beyond the offending damage. Dysregulation of this DDT pathway in human cells leads to increased mutations rates that may contribute to the onset of cancer. Furthermore, TLS affords human cancer cells the ability to counteract chemotherapeutic agents that elicit cell death by damaging DNA in actively replicating cells. Currently, it is unclear how this critical pathway unfolds, in particular where and when TLS occurs on each template strand. Given the semi-discontinuous nature of DNA replication, it is likely that TLS on the leading and lagging strand templates is unique for each strand. Since the discovery of DDT in the late 1960’s, most studies on TLS in eukaryotes have focused on DNA lesions resulting from ultraviolet (UV) radiation exposure. In this review, we re-visit these and other related studies to dissect the step-by-step intricacies of this complex process, provide our current understanding of TLS on leading and lagging strand templates, and propose testable hypotheses to gain further insights.

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Structure and mechanism of human PrimPol, a DNA polymerase with primase activity

Abstract: Analysis of crystal structure elucidates the mechanism by which a human enzyme acts as both a primase and a DNA polymerase.

Cited by 70 publications

References 44 publications

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

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

Translesion and Repair DNA Polymerases: Diverse Structure and Mechanism

Eukaryotic Translesion DNA Synthesis on the Leading and Lagging Strands: Unique Detours around the Same Obstacle

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