PrimPol is required for replication reinitiation after mtDNA damage

Torregrosa‐Muñumer, Rubén; Forslund, Josefin M. E.; Goffart, Steffi; Pfeiffer, Annika; Stojkovič, Gorazd; Carvalho, Gustavo; Al-Furoukh, Natalie; Blanco, Luis; Wanrooij, Sjoerd; Pohjoismäki, Jaakko L. O.

doi:10.1073/pnas.1705367114

Cited by 79 publications

(99 citation statements)

References 58 publications

(69 reference statements)

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“…1G). This may suggest changes in the mode of replication or initiation as this region contains the origins of replication and transcription, similar to a previous study on PrimPol -/-MEFs that showed replication changes in this region (24).…”

Section: Primpol -/Cells Have a Defect In Mitochondrial Dna Proliferasupporting

confidence: 88%

“…In addition, we also found a similar increase (~ 51%) in mtDNA copy number in DT40 PrimPol -/cells, which could be complemented by the addition of the WT protein ( Fig. S3B), whilst similar increases have also been reported in MEFs (24). Upon staining these cells with an anti-DNA antibody, we found a clear increase in mtDNA nucleoid number but no increase in nucleoid size (Fig.…”

Section: Primpol -/Cells Have a Defect In Mitochondrial Dna Proliferasupporting

confidence: 82%

“…As well as nuclear DNA maintenance, PrimPol is also found in mitochondria (mt) where it is involved in the replication of mtDNA (11,23,24). Unlike in the nucleus, human mitochondria contain multiple copies of a 16 kb circular DNA molecule organised into nucleoids that encodes 13 components of the electron transport chain, 22 tRNAs and 2 rRNAs.…”

Section: Introductionmentioning

confidence: 99%

“…mtDNA is replicated by a dedicated polymerase, Pol γ, and a range of other repair and replication proteins are utilised, some of which are mitochondrial-specific and others have dual roles in both the nucleus and mitochondrion (23,(25)(26)(27)(28). PrimPol has been reported to be important for repriming of mtDNA replication after damage (24), but little is still known about its recruitment to the organelle or its DNA, although it has been shown to interact with mtSSB in a manner functionally similar to RPA (29). Thus, PrimPol is a dynamic protein that likely fulfils similar roles in DNA replication in both the nuclear and mitochondrial compartments.…”

Section: Introductionmentioning

confidence: 99%

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PrimPol is required for the maintenance of efficient nuclear and mitochondrial DNA replication in human cells

Bailey

Bianchi

Doherty

2018

Preprint

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Eukaryotic Primase-Polymerase (PrimPol) is an enzyme that maintains efficient DNA duplication by repriming replication restart downstream of replicase stalling lesions and structures. To elucidate the cellular requirements for PrimPol in human cells, we generated PrimPol-deleted cell lines and show that it plays key roles in maintaining active replication in both the nucleus and mitochondrion, even in the absence of exogenous damage. Human cells lacking PrimPol exhibit delayed recovery after UV-C damage and increased mutation rates, micronuclei and sister chromatin exchanges but are not sensitive to genotoxins. PrimPol is also required during mitochondrial replication, with PrimPol-deficient cells having increased mtDNA copy number but displaying a significant decrease in replication rates. Deletion of PrimPol in XPV cells, lacking functional Pol Eta, causes an increase in DNA damage sensitivity and pronounced fork stalling after UV-C treatment. We show that, unlike canonical TLS polymerases, PrimPol is important for allowing active replication to proceed, even in the absence of exogenous damage, thus preventing the accumulation of excessive fork stalling and genetic mutations. Together, these findings highlight the importance of PrimPol for maintaining efficient DNA replication in unperturbed cells and its complementary roles, with Pol Eta, in damage tolerance in human cells.(200 words)

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Section: Primpol -/Cells Have a Defect In Mitochondrial Dna Proliferasupporting

confidence: 88%

Section: Primpol -/Cells Have a Defect In Mitochondrial Dna Proliferasupporting

confidence: 82%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

PrimPol is required for the maintenance of efficient nuclear and mitochondrial DNA replication in human cells

Bailey

Bianchi

Doherty

2018

Preprint

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“…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).…”

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confidence: 99%

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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Ribonucleotides in mitochondrial DNA

Wanrooij

Chabes

2019

FEBS Letters

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The incorporation of ribonucleotides (rNMPs) into DNA during genome replication has gained substantial attention in recent years and has been shown to be a significant source of genomic instability. Studies in yeast and mammals have shown that the two genomes, the nuclear DNA (nDNA) and the mitochondrial DNA (mtDNA), differ with regard to their rNMP content. This is largely due to differences in rNMP repair – whereas rNMPs are efficiently removed from the nuclear genome, mitochondria lack robust mechanisms for removal of single rNMPs incorporated during DNA replication. In this minireview, we describe the processes that determine the frequency of rNMPs in the mitochondrial genome and summarise recent findings regarding the effect of incorporated rNMPs on mtDNA stability and function.

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PrimPol is required for replication reinitiation after mtDNA damage

Cited by 79 publications

References 58 publications

PrimPol is required for the maintenance of efficient nuclear and mitochondrial DNA replication in human cells

PrimPol is required for the maintenance of efficient nuclear and mitochondrial DNA replication in human cells

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

Ribonucleotides in mitochondrial DNA

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