The role of catalytic and regulatory domains of human PrimPol in DNA binding and synthesis.

Human PrimPol possesses DNA primase and DNA polymerase activities and restarts stalled replication forks protecting cells against DNA damage in nuclei and mitochondria. The zinc-binding motif (ZnFn) of the C-terminal domain (CTD) of PrimPol is required for DNA primase activity but the mechanism is not clear. In this work, we biochemically demonstrate that PrimPol initiates de novo DNA synthesis in cis-orientation, when the N-terminal catalytic domain (NTD) and the CTD of the same molecule cooperate for substrates binding and catalysis. The modeling studies revealed that PrimPol uses a similar mode of initiating NTP coordination as the human primase. The ZnFn motif residue Arg417 is required for binding the 5'-triphosphate group that stabilizes the PrimPol complex with a DNA template-primer. We found that the NTD alone is able to initiate DNA synthesis, and the CTD stimulates the primase activity of NTD. The regulatory role of the RPA-binding motif in the modulation of PrimPol binding to DNA is also demonstrated.

8-Oxoadenine: A «New¼ Player of the Oxidative Stress in Mammals?

Numerous studies have shown that oxidative modifications of guanine (7,8-dihydro-8-oxoguanine, 8-oxoG) can affect cellular functions. 7,8-Dihydro-8-oxoadenine (8-oxoA) is another abundant paradigmatic ambiguous nucleobase but findings reported on the mutagenicity of 8-oxoA in bacterial and eukaryotic cells are incomplete and contradictory. Although several genotoxic studies have demonstrated the mutagenic potential of 8-oxoA in eukaryotic cells, very little biochemical and bioinformatics data about the mechanism of 8-oxoA-induced mutagenesis are available. In this review, we discuss dual coding properties of 8-oxoA, summarize historical and recent genotoxicity and biochemical studies, and address the main protective cellular mechanisms of response to 8-oxoA. We also discuss the available structural data for 8-oxoA bypass by different DNA polymerases as well as the mechanisms of 8-oxoA recognition by DNA repair enzymes.

The Catalytic Activity of Human REV1 on Undamaged and Damaged DNA.

Eukaryotic REV1 serves as a scaffold protein for the coordination of DNA polymerases during DNA translesion synthesis. Besides this structural role, REV1 is a Y-family DNA polymerase with its own distributive deoxycytidyl transferase activity. However, data about the accuracy and efficiency of DNA synthesis by REV1 in the literature are contrasting. Here, we expressed and purified the full-length human REV1 from Saccharomyces cerevisiae and characterized its activity on undamaged DNA and a wide range of damaged DNA templates. We demonstrated that REV1 carried out accurate synthesis opposite 8-oxoG and O6-meG with moderate efficiency. It also replicated thymine glycol surprisingly well in an error-prone manner, but was blocked by the intrastrand 1,2-GG cisplatin crosslink. By using the 1,N6-ethenoadenine and 7-deaza-adenine lesions, we have provided biochemical evidence of the importance for REV1 functioning of the Hoogsteen face of template A, the second preferable template after G.

Regulation of Human DNA Primase-Polymerase PrimPol.

Transmission of genetic information depends on successful completion of DNA replication. Genomic DNA is subjected to damage on a daily basis. DNA lesions create obstacles for DNA polymerases and can lead to the replication blockage, formation of DNA breaks, cell cycle arrest, and apoptosis. Cells have evolutionary adapted to DNA damage by developing mechanisms allowing elimination of lesions prior to DNA replication (DNA repair) and helping to bypass lesions during DNA synthesis (DNA damage tolerance). The second group of mechanisms includes the restart of DNA synthesis at the sites of DNA damage by DNA primase-polymerase PrimPol. Human PrimPol was described in 2013. The properties and functions of this enzyme have been extensively studied in recent years, but very little is known about the regulation of PrimPol and association between the enzyme dysfunction and diseases. In this review, we described the mechanisms of human PrimPol regulation in the context of DNA replication, discussed in detail interactions of PrimPol with other proteins, and proposed possible pathways for the regulation of human PrimPol activity. The article also addresses the association of PrimPol dysfunction with human diseases.

Response of PRIMPOL-Knockout Human Lung Adenocarcinoma A549 Cells to Genotoxic Stress.

Human DNA primase/polymerase PrimPol synthesizes DNA primers de novo after replication fork stalling at the sites of DNA damage, thus contributing to the DNA damage tolerance. The role of PrimPol in response to the different types of DNA damage is poorly understood. We knocked out the PRIMPOL gene in the lung carcinoma A549 cell line and characterized the response of the obtained cells to the DNA damage caused by hydrogen peroxide, methyl methanesulfonate (MMS), cisplatin, bleomycin, and ionizing radiation. The PRIMPOL knockout reduced the number of proliferating cells and cells in the G2 phase after treatment with MMS and caused a more pronounced delay of the S phase in the cisplatin-treated cells. Ionizing radiation at a dose of 10 Gy significantly increased the content of apoptotic cells among the PRIMPOL-deficient cells, while the proportion of cells undergoing necroptosis increased in both parental and knockout cells at any radiation dose. The viability of PRIMPOL-deficient cells upon the hydrogen peroxide-induced oxidative stress increased compared to the control cells, as determined by the methyl tetrazolium (MTT) assay. The obtained data indicate the involvement of PRIMPOL in the modulation of adaptive cell response to various types of genotoxic stress.

Multifaceted Nature of DNA Polymerase θ.

DNA polymerase θ belongs to the A family of DNA polymerases and plays a key role in DNA repair and damage tolerance, including double-strand break repair and DNA translesion synthesis. Pol θ is often overexpressed in cancer cells and promotes their resistance to chemotherapeutic agents. In this review, we discuss unique biochemical properties and structural features of Pol θ, its multiple roles in protection of genome stability and the potential of Pol θ as a target for cancer treatment.

Bypass of Abasic Site-Peptide Cross-Links by Human Repair and Translesion DNA Polymerases.

DNA-protein cross-links remain the least-studied type of DNA damage. Recently, their repair was shown to involve proteolysis; however, the fate of the peptide remnant attached to DNA is unclear. Particularly, peptide cross-links could interfere with DNA polymerases. Apurinuic/apyrimidinic (AP) sites, abundant and spontaneously arising DNA lesions, readily form cross-links with proteins. Their degradation products (AP site-peptide cross-links, APPXLs) are non-instructive and should be even more problematic for polymerases. Here, we address the ability of human DNA polymerases involved in DNA repair and translesion synthesis (POLß, POLλ, POLη, POLκ and PrimPOL) to carry out synthesis on templates containing AP sites cross-linked to the N-terminus of a 10-mer peptide (APPXL-I) or to an internal lysine of a 23-mer peptide (APPXL-Y). Generally, APPXLs strongly blocked processive DNA synthesis. The blocking properties of APPXL-I were comparable with those of an AP site, while APPXL-Y constituted a much stronger obstruction. POLη and POLκ demonstrated the highest bypass ability. DNA polymerases mostly used dNTP-stabilized template misalignment to incorporate nucleotides when encountering an APPXL. We conclude that APPXLs are likely highly cytotoxic and mutagenic intermediates of AP site-protein cross-link repair and must be quickly eliminated before replication.

DNA Polymerase and dRP-lyase activities of polymorphic variants of human Pol ι.

Y-family DNA polymerase iota (Pol ι) is involved in DNA damage response and tolerance. Mutations and altered expression level of POLI gene are linked to a higher incidence of cancer. We biochemically characterized five active site polymorphic variants of human Pol ι: R71G (rs3218778), P118L (rs554252419), I236M (rs3218784), E251K (rs3218783) and P365R (rs200852409). We analyzed fidelity of nucleotide incorporation on undamaged DNA, efficiency and accuracy of DNA damage bypass, as well as 5'-deoxyribophosphate lyase (dRP-lyase) activity. The I236M and P118L variants were indistinguishable from the wild-type Pol ι in activity. The E251K and P365R substitutions altered the spectrum of nucleotide incorporation opposite several undamaged DNA bases. The P365R variant also reduced the dRP-lyase activity and possessed the decreased TLS activity opposite 8-oxo-G. The R71G mutation dramatically affected the catalytic activities of Pol ι. The reduced DNA polymerase activity of the R71G variant correlated with an enhanced fidelity of nucleotide incorporation on undamaged DNA, altered lesion-bypass activity and reduced dRP-lyase activity. Therefore, this amino acid substitution likely alters Pol ι functions in vivo.

A Multifunctional Protein PolDIP2 in DNA Translesion Synthesis.

Polymerase δ-interacting protein 2 (PolDIP2) is involved in the multiple protein-protein interactions and plays roles in many cellular processes including regulation of the nuclear redox environment, organization of the mitotic spindle and chromosome segregation, pre-mRNA processing, mitochondrial morphology and functions, cell migration and cellular adhesion. PolDIP2 is also a binding partner of high-fidelity DNA polymerase delta, PCNA and a number of translesion and repair DNA polymerases. The growing evidence suggests that PolDIP2 is a general regulatory protein in DNA damage response. However PolDIP2 functions in DNA translesion synthesis and repair are not fully understood. In this review, we address the functional interaction of PolDIP2 with human DNA polymerases and discuss the possible functions in DNA damage response.

Strand Displacement Activity of PrimPol.

Human PrimPol is a unique enzyme possessing DNA/RNA primase and DNA polymerase activities. In this work, we demonstrated that PrimPol efficiently fills a 5-nt gap and possesses the conditional strand displacement activity stimulated by Mn2+ ions and accessory replicative proteins RPA and PolDIP2. The DNA displacement activity of PrimPol was found to be more efficient than the RNA displacement activity and FEN1 processed the 5'-DNA flaps generated by PrimPol in vitro.

Yeast DNA polymerase ζ maintains consistent activity and mutagenicity across a wide range of physiological dNTP concentrations.

In yeast, dNTP pools expand drastically during DNA damage response. We show that similar dNTP elevation occurs in strains, in which intrinsic replisome defects promote the participation of error-prone DNA polymerase ζ (Polζ) in replication of undamaged DNA. To understand the significance of dNTP pools increase for Polζ function, we studied the activity and fidelity of four-subunit Polζ (Polζ4) and Polζ4-Rev1 (Polζ5) complexes in vitro at 'normal S-phase' and 'damage-response' dNTP concentrations. The presence of Rev1 inhibited the activity of Polζ and greatly increased the rate of all three 'X-dCTP' mispairs, which Polζ4 alone made extremely inefficiently. Both Polζ4 and Polζ5 were most promiscuous at G nucleotides and frequently generated multiple closely spaced sequence changes. Surprisingly, the shift from 'S-phase' to 'damage-response' dNTP levels only minimally affected the activity, fidelity and error specificity of Polζ complexes. Moreover, Polζ-dependent mutagenesis triggered by replisome defects or UV irradiation in vivo was not decreased when dNTP synthesis was suppressed by hydroxyurea, indicating that Polζ function does not require high dNTP levels. The results support a model wherein dNTP elevation is needed to facilitate non-mutagenic tolerance pathways, while Polζ synthesis represents a unique mechanism of rescuing stalled replication when dNTP supply is low.

Isoforms of Base Excision Repair Enzymes Produced by Alternative Splicing.

Transcripts of many enzymes involved in base excision repair (BER) undergo extensive alternative splicing, but functions of the corresponding alternative splice variants remain largely unexplored. In this review, we cover the studies describing the common alternatively spliced isoforms and disease-associated variants of DNA glycosylases, AP-endonuclease 1, and DNA polymerase beta. We also discuss the roles of alternative splicing in the regulation of their expression, catalytic activities, and intracellular transport.

The Dimeric Architecture of Checkpoint Kinases Mec1ATR and Tel1ATM Reveal a Common Structural Organization.

The phosphatidylinositol 3-kinase-related protein kinases are key regulators controlling a wide range of cellular events. The yeast Tel1 and Mec1·Ddc2 complex (ATM and ATR-ATRIP in humans) play pivotal roles in DNA replication, DNA damage signaling, and repair. Here, we present the first structural insight for dimers of Mec1·Ddc2 and Tel1 using single-particle electron microscopy. Both kinases reveal a head to head dimer with one major dimeric interface through the N-terminal HEAT (named after Huntingtin, elongation factor 3, protein phosphatase 2A, and yeast kinase TOR1) repeat. Their dimeric interface is significantly distinct from the interface of mTOR complex 1 dimer, which oligomerizes through two spatially separate interfaces. We also observe different structural organizations of kinase domains of Mec1 and Tel1. The kinase domains in the Mec1·Ddc2 dimer are located in close proximity to each other. However, in the Tel1 dimer they are fully separated, providing potential access of substrates to this kinase, even in its dimeric form.

DNA Damage Tolerance by Eukaryotic DNA Polymerase and Primase PrimPol.

PrimPol is a human deoxyribonucleic acid (DNA) polymerase that also possesses primase activity and is involved in DNA damage tolerance, the prevention of genome instability and mitochondrial DNA maintenance. In this review, we focus on recent advances in biochemical and crystallographic studies of PrimPol, as well as in identification of new protein-protein interaction partners. Furthermore, we discuss the possible functions of PrimPol in both the nucleus and the mitochondria.

Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua.

Hepatocellular carcinomas (HCCs) are the third leading cause of cancer deaths worldwide. The highest rates of early onset HCCs occur in geographical regions with high aflatoxin B1 (AFB1) exposure, concomitant with hepatitis B infection. Although the carcinogenic basis of AFB1 has been ascribed to its mutagenic effects, the mutagenic property of the primary AFB1-DNA adduct, AFB1-N7-Gua, in mammalian cells has not been studied extensively. Taking advantage of the ability to create vectors containing a site-specific DNA adduct, the mutagenic potential was determined in primate cells. This adduct was highly mutagenic following replication in COS-7 cells, with a mutation frequency of 45%. The spectrum of mutations was predominantly G to T base substitutions, a result that is consistent with previous mutation data derived from aflatoxin-associated HCCs. To assess which DNA polymerases (pol) might contribute to the mutational outcome, in vitro replication studies were performed. Unexpectedly, replicative pol δ and the error-prone translesion synthesis pol ζ were able to accurately bypass AFB1-N7-Gua. In contrast, replication bypass using pol κ was shown to occur with low fidelity and could account for the commonly detected G to T transversions.

Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct.

Aflatoxin B1 (AFB1) is a known carcinogen associated with early-onset hepatocellular carcinoma (HCC) and is thought to contribute to over half a million new HCCs per year. Although some of the fundamental risk factors are established, the molecular basis of AFB1-induced mutagenesis in primate cells has not been rigorously investigated. To gain insights into genome instability that is produced as a result of replicating DNAs containing AFB1 adducts, site-specific mutagenesis assays were used to establish the mutagenic potential of the persistent ring-opened AFB1 adduct, AFB1-formamidopyrimidine (AFB1-FAPY). This lesion was highly mutagenic, yielding replication error frequencies of 97%, with the predominant base substitution being a G to T transversion. This transversion is consistent with previous mutational data derived from aflatoxin-associated HCCs. In vitro translesion synthesis assays demonstrated that polymerase (pol) ζ was the most likely candidate polymerase that is responsible for the G to T mutations induced by this adduct.

A four-subunit DNA polymerase ζ complex containing Pol δ accessory subunits is essential for PCNA-mediated mutagenesis.

DNA polymerase ζ (Pol ζ) plays a key role in DNA translesion synthesis (TLS) and mutagenesis in eukaryotes. Previously, a two-subunit Rev3-Rev7 complex had been identified as the minimal assembly required for catalytic activity in vitro. Herein, we show that Saccharomyces cerevisiae Pol ζ binds to the Pol31 and Pol32 subunits of Pol δ, forming a four-subunit Pol ζ(4) complex (Rev3-Rev7-Pol31-Pol32). A [4Fe-4S] cluster in Rev3 is essential for the formation of Pol ζ(4) and damage-induced mutagenesis. Pol32 is indispensible for complex formation, providing an explanation for the long-standing observation that pol32Δ strains are defective for mutagenesis. The Pol31 and Pol32 subunits are also required for proliferating cell nuclear antigen (PCNA)-dependent TLS by Pol ζ as Pol ζ(2) lacks functional interactions with PCNA. Mutation of the C-terminal PCNA-interaction motif in Pol32 attenuates PCNA-dependent TLS in vitro and mutagenesis in vivo. Furthermore, a mutant form of PCNA, encoded by the mutagenesis-defective pol30-113 mutant, fails to stimulate Pol ζ(4) activity, providing an explanation for the observed mutagenesis phenotype. A stable Pol ζ(4) complex can be identified in all phases of the cell cycle suggesting that this complex is not regulated at the level of protein interactions between Rev3-Rev7 and Pol31-Pol32.

DNA synthesis across DNA hairpins by human PrimPol.

PrimPol is a human DNA primase involved in DNA damage tolerance pathways by restarting DNA replication downstream of DNA lesions and non-canonical DNA structures. Activity and affinity to DNA relays on the interaction of PrimPol with replication protein A (RPA). In this work, we report that PrimPol has an intrinsic ability to copy DNA hairpins with a stem length of 5-9 base pairs (bp) but shows pronounced pausing of DNA synthesis. RPA greatly stimulates DNA synthesis across inverted DNA repeats by PrimPol. Moreover, deletion of the C-terminal RPA binding motif (RBM) facilitates DNA hairpin bypass and makes it independent of RPA. This work supports the idea that RBM is a negative regulator of PrimPol and its interaction with RPA is required to achieve the fully active state.

PrimPol Variant V102A with Altered Primase and Polymerase Activities.

PrimPol is a human DNA primase-polymerase which restarts DNA synthesis beyond DNA lesions and non-B DNA structures blocking replication. Disfunction of PrimPol in cells leads to slowing of DNA replication rates in mitochondria and nucleus, accumulation of chromosome aberrations, cell cycle delay, and elevated sensitivity to DNA-damaging agents. A defective PrimPol has been suggested to be associated with the development of ophthalmic diseases, elevated mitochondrial toxicity of antiviral drugs and increased cell resistance to chemotherapy. Here, we describe a rare missense PrimPol variant V102A with altered biochemical properties identified in patients suffering from ovarian and cervical cancer. The Val102 to Ala substitution dramatically reduced both the primase and DNA polymerase activities of PrimPol as well as specifically decreased its ability to incorporate ribonucleotides. Structural analysis indicates that the V102A substitution can destabilize the hydrophobic pocket adjacent to the active site, affecting dNTP binding and catalysis.

Methylation and hydroxymethylation of cytosine alter activity and fidelity of translesion DNA polymerases.

Epigenetic cytosine methylation covers most of genomic CpG dinucleotides in human cells. In addition to common deamination-mediated mutagenesis at CpG sites, an alternative deamination-independent pathway associated with DNA polymerase activity was previously described. This mutagenesis is characterized by the TCG→TTG mutational signature and is believed to arise from dAMP misincorporation opposite 5-methylcytosine (mC) or its oxidized derivative 5-hydroxymethylcytosine (hmC) by B-family replicative DNA polymerases with disrupted proofreading 3→5'-exonuclease activity. In addition to being less stable and pro-mutagenic themselves, cytosine modifications also increase the risk of adjacent nucleotides damage, including the formation of 8-oxo-2'-deoxyguanosine (8-oxoG), a well-known mutagenic lesion. The effect of cytosine methylation on error-prone DNA polymerases lacking proofreading activity and involved in repair and DNA translesion synthesis remains unexplored. Here we analyze the efficiency and fidelity of translesion Y-family polymerases (Pol κ, Pol η, Pol ι and REV1) and primase-polymerase PrimPol opposite mC and hmC as well as opposite 8-oxoG adjacent to mC in the TCG context. We demonstrate that epigenetic cytosine modifications suppress Pol ι and REV1 activities and lead to increasing dAMP misincorporation by PrimPol, Pol κ and Pol ι in vitro. Cytosine methylation also increases misincorporation of dAMP opposite the adjacent 8-oxoG by PrimPol, decreases the TLS activity of Pol η opposite the lesion but increases dCMP incorporation opposite 8-oxoG by REV1. Altogether, these data suggest that methylation and hydroxymethylation of cytosine alter activity and fidelity of translesion DNA polymerases.