Human primosome requires replication protein A when copying DNA with inverted repeats.

The human primosome, a four-subunit complex of primase and DNA polymerase alpha (Polα), initiates DNA synthesis on both chromosome strands by generating chimeric RNA-DNA primers for loading DNA polymerases delta and epsilon (Polε). Replication protein A (RPA) tightly binds to single-stranded DNA strands, protecting them from nucleolytic digestion and unauthorized transactions. We report here that RPA plays a critical role for the human primosome during DNA synthesis across inverted repeats prone to hairpin formation. On other alternatively structured DNA forming a G-quadruplex, RPA provides no assistance for primosome. A stimulatory effect of RPA on DNA synthesis across hairpins was also observed for the catalytic domain of Polα but not of Polε. The important factors for an efficient hairpin bypass by primosome are the high affinity of RPA to DNA based on four DNA-binding domains and the interaction of the winged-helix-turn-helix domain of RPA with Polα. Binding studies indicate that this interaction stabilizes the RPA/Polα complex on the primed template. This work provides insight into a cooperative action of RPA and primosome on DNA, which is critical for DNA synthesis across inverted repeats.

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.

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.

DNA primase large subunit is an essential plant gene for geminiviruses, putatively priming viral ss-DNA replication.

The family of Geminiviridae consists of more than 500 circular single-stranded (ss) DNA viral species that can infect numerous dicot and monocot plants. Geminiviruses replicate their genome in the nucleus of a plant cell, taking advantage of the host's DNA replication machinery. For converting their DNA into double-stranded DNA, and subsequent replication, these viruses rely on host DNA polymerases. However, the priming of the very first step of this process, i.e. the conversion of incoming circular ssDNA into a dsDNA molecule, has remained elusive for almost 30 years. In this study, sequencing of melon (Cucumis melo) accession K18 carrying the Tomato leaf curl New Delhi virus (ToLCNDV) recessive resistance quantitative trait locus (QTL) in chromosome 11, and analyses of DNA sequence data from 100 melon genomes, showed a conservation of a shared mutation in the DNA Primase Large subunit (PRiL) of all accessions that exhibited resistance upon a challenge with ToLCNDV. Silencing of (native) Nicotiana benthamiana PriL and subsequent challenging with three different geminiviruses showed a severe reduction in titers of all three viruses, altogether emphasizing an important role of PRiL in geminiviral replication. A model is presented explaining the role of PriL during initiation of geminiviral DNA replication, i.e. as a regulatory subunit of primase that generates an RNA primer at the onset of DNA replication in analogy to DNA Primase-mediated initiation of DNA replication in all living organisms.

Structures of human primosome elongation complexes.

The synthesis of RNA-DNA primer by primosome requires coordination between primase and DNA polymerase α subunits, which is accompanied by unknown architectural rearrangements of multiple domains. Using cryogenic electron microscopy, we solved a 3.6 Å human primosome structure caught at an early stage of RNA primer elongation with deoxynucleotides. The structure confirms a long-standing role of primase large subunit and reveals new insights into how primosome is limited to synthesizing short RNA-DNA primers.

Human DNA polymerase α has a strong mutagenic potential at the initial steps of DNA synthesis.

DNA polymerase α (Polα) is essential for DNA replication initiation and makes a notable contribution to genome mutagenesis. The activity and fidelity of Polα during the early steps of DNA replication have not been well studied. Here we show that at the beginning of DNA synthesis, when extending the RNA primer received from primase, Polα is more mutagenic than during the later DNA elongation steps. Kinetic and binding studies revealed substantially higher activity and affinity to the template:primer when Polα interacts with ribonucleotides of a chimeric RNA-DNA primer. Polα activity greatly varies during first six steps of DNA synthesis, and the bias in the rates of correct and incorrect dNTP incorporation leads to impaired fidelity, especially upon the second step of RNA primer extension. Furthermore, increased activity and stability of Polα/template:primer complexes containing RNA-DNA primers result in higher efficiency of mismatch extension.

The iron-sulfur cluster is essential for DNA binding by human DNA polymerase ε.

DNA polymerase ε (Polε) is a key enzyme for DNA replication in eukaryotes. Recently it was shown that the catalytic domain of yeast Polε (PolεCD) contains a [4Fe-4S] cluster located at the base of the processivity domain (P-domain) and coordinated by four conserved cysteines. In this work, we show that human PolεCD (hPolεCD) expressed in bacterial cells also contains an iron-sulfur cluster. In comparison, recombinant hPolεCD produced in insect cells contains significantly lower level of iron. The iron content of purified hPolECD samples correlates with the level of DNA-binding molecules, which suggests an important role of the iron-sulfur cluster in hPolε interaction with DNA. Indeed, mutation of two conserved cysteines that coordinate the cluster abolished template:primer binding as well as DNA polymerase and proofreading exonuclease activities. We propose that the cluster regulates the conformation of the P-domain, which, like a gatekeeper, controls access to a DNA-binding cleft for a template:primer. The binding studies demonstrated low affinity of hPolεCD to DNA and a strong effect of salt concentration on stability of the hPolεCD/DNA complex. Pre-steady-state kinetic studies have shown a maximal polymerization rate constant of 51.5 s-1 and a relatively low affinity to incoming dNTP with an apparent KD of 105 µM.

Insight into RNA-DNA primer length counting by human primosome.

The human primosome, a four-subunit complex of primase and DNA polymerase alpha (Polα), synthesizes chimeric RNA-DNA primers of a limited length for DNA polymerases delta and epsilon to initiate DNA replication on both chromosome strands. Despite recent structural insights into the action of its two catalytic centers, the mechanism of DNA synthesis termination is still unclear. Here we report results of functional and structural studies revealing how the human primosome counts RNA-DNA primer length and timely terminates DNA elongation. Using a single-turnover primer extension assay, we defined two factors that determine a mature primer length (∼35-mer): (i) a tight interaction of the C-terminal domain of the DNA primase large subunit (p58C) with the primer 5'-end, and (ii) flexible tethering of p58C and the DNA polymerase alpha catalytic core domain (p180core) to the primosome platform domain by extended linkers. The obtained data allow us to conclude that p58C is a key regulator of all steps of RNA-DNA primer synthesis. The above-described findings provide a notable insight into the mechanism of DNA synthesis termination by a eukaryotic primosome, an important process for ensuring successful primer handover to replication DNA polymerases and for maintaining genome integrity.

Efficient discrimination against RNA-containing primers by human DNA polymerase ε.

DNA polymerase ε (Polε) performs bulk synthesis of DNA on the leading strand during genome replication. Polε binds two substrates, a template:primer and dNTP, and catalyzes a covalent attachment of dNMP to the 3' end of the primer. Previous studies have shown that Polε easily inserts and extends ribonucleotides, which may promote mutagenesis and genome instability. In this work, we analyzed the mechanisms of discrimination against RNA-containing primers by human Polε (hPolε), performing binding and kinetic studies at near-physiological salt concentration. Pre-steady-state kinetic studies revealed that hPolεCD extends RNA primers with approximately 3300-fold lower efficiency in comparison to DNA, and addition of one dNMP to the 3' end of an RNA primer increases activity 36-fold. Likewise, addition of one rNMP to the 3' end of a DNA primer reduces activity 38-fold. The binding studies conducted in the presence of 0.15 M NaCl revealed that human hPolεCD has low affinity to DNA (KD of 1.5 µM). Strikingly, a change of salt concentration from 0.1 M to 0.15 M reduces the stability of the hPolεCD/DNA complex by 25-fold. Upon template:primer binding, the incoming dNTP and magnesium ions make hPolε discriminative against RNA and chimeric RNA-DNA primers. In summary, our studies revealed that hPolε discrimination against RNA-containing primers is based on the following factors: incoming dNTP, magnesium ions, a steric gate for the primer 2'OH, and the rigid template:primer binding pocket near the catalytic site. In addition, we showed the importance of conducting functional studies at near-physiological salt concentration.

Structural and functional insight into mismatch extension by human DNA polymerase α.

Human DNA polymerase α (Polα) does not possess proofreading ability and plays an important role in genome replication and mutagenesis. Polα extends the RNA primers generated by primase and provides a springboard for loading other replication factors. Here we provide the structural and functional analysis of the human Polα interaction with a mismatched template:primer. The structure of the human Polα catalytic domain in the complex with an incoming deoxycytidine triphosphate (dCTP) and the template:primer containing a T-C mismatch at the growing primer terminus was solved at a 2.9 Å resolution. It revealed the absence of significant distortions in the active site and in the conformation of the substrates, except the primer 3'-end. The T-C mismatch acquired a planar geometry where both nucleotides moved toward each other by 0.4 Å and 0.7 Å, respectively, and made one hydrogen bond. The binding studies conducted at a physiological salt concentration revealed that Polα has a low affinity to DNA and is not able to discriminate against a mispaired template:primer in the absence of deoxynucleotide triphosphate (dNTP). Strikingly, in the presence of cognate dNTP, Polα showed a more than 10-fold higher selectivity for a correct duplex versus a mismatched one. According to pre-steady-state kinetic studies, human Polα extends the T-C mismatch with a 249-fold lower efficiency due to reduction of the polymerization rate constant by 38-fold and reduced affinity to the incoming nucleotide by 6.6-fold. Thus, a mismatch at the postinsertion site affects all factors important for primer extension: affinity to both substrates and the rate of DNA polymerization.

Translesion activity of PrimPol on DNA with cisplatin and DNA-protein cross-links.

Human PrimPol belongs to the archaeo-eukaryotic primase superfamily of primases and is involved in de novo DNA synthesis downstream of blocking DNA lesions and non-B DNA structures. PrimPol possesses both DNA/RNA primase and DNA polymerase activities, and also bypasses a number of DNA lesions in vitro. In this work, we have analyzed translesion synthesis activity of PrimPol in vitro on DNA with an 1,2-intrastrand cisplatin cross-link (1,2-GG CisPt CL) or a model DNA-protein cross-link (DpCL). PrimPol was capable of the 1,2-GG CisPt CL bypass in the presence of Mn2+ ions and preferentially incorporated two complementary dCMPs opposite the lesion. Nucleotide incorporation was stimulated by PolDIP2, and yeast Pol ζ efficiently extended from the nucleotides inserted opposite the 1,2-GG CisPt CL in vitro. DpCLs significantly blocked the DNA polymerase activity and strand displacement synthesis of PrimPol. However, PrimPol was able to reach the DpCL site in single strand template DNA in the presence of both Mg2+ and Mn2+ ions despite the presence of the bulky protein obstacle.

Replication protein A binds RNA and promotes R-loop formation.

Replication protein A (RPA), a major eukaryotic ssDNA-binding protein, is essential for all metabolic processes that involve ssDNA, including DNA replication, repair, and damage signaling. To perform its functions, RPA binds ssDNA tightly. In contrast, it was presumed that RPA binds RNA weakly. However, recent data suggest that RPA may play a role in RNA metabolism. RPA stimulates RNA-templated DNA repair in vitro and associates in vivo with R-loops, the three-stranded structures consisting of an RNA-DNA hybrid and the displaced ssDNA strand. R-loops are common in the genomes of pro- and eukaryotes, including humans, and may play an important role in transcription-coupled homologous recombination and DNA replication restart. However, the mechanism of R-loop formation remains unknown. Here, we investigated the RNA-binding properties of human RPA and its possible role in R-loop formation. Using gel-retardation and RNA/DNA competition assays, we found that RPA binds RNA with an unexpectedly high affinity (KD ≈ 100 pm). Furthermore, RPA, by forming a complex with RNA, can promote R-loop formation with homologous dsDNA. In reconstitution experiments, we showed that human DNA polymerases can utilize RPA-generated R-loops for initiation of DNA synthesis, mimicking the process of replication restart in vivo These results demonstrate that RPA binds RNA with high affinity, supporting the role of this protein in RNA metabolism and suggesting a mechanism of genome maintenance that depends on RPA-mediated DNA replication restart.

Iron-Sulfur Clusters in DNA Polymerases and Primases of Eukaryotes.

Research during the past decade witnessed the discovery of [4Fe-4S] clusters in several members of the eukaryotic DNA replication machinery. The presence of clusters was confirmed by UV-visible absorption, electron paramagnetic resonance spectroscopy, and metal analysis for primase and the B-family DNA polymerases δ and ζ. The crystal structure of primase revealed that the [4Fe-4S] cluster is buried inside the protein and fulfills a structural role. Although [4Fe-4S] clusters are firmly established in the C-terminal domains of catalytic subunits of DNA polymerases δ and ζ, no structures are currently available and their precise roles have not been ascertained. The [4Fe-4S] clusters in the polymerases and primase play a structural role ensuring proper protein folding and stability. In DNA polymerases δ and ζ, they can potentially play regulatory role by sensing hurdles during DNA replication and assisting with DNA polymerase switches by oscillation between oxidized-reduced states.

Activity and fidelity of human DNA polymerase α depend on primer structure.

DNA polymerase α (Polα) plays an important role in genome replication. In a complex with primase, Polα synthesizes chimeric RNA-DNA primers necessary for replication of both chromosomal DNA strands. During RNA primer extension with deoxyribonucleotides, Polα needs to use double-stranded helical substrates having different structures. Here, we provide a detailed structure-function analysis of human Polα's interaction with dNTPs and DNA templates primed with RNA, chimeric RNA-DNA, or DNA. We report the crystal structures of two ternary complexes of the Polα catalytic domain containing dCTP, a DNA template, and either a DNA or an RNA primer. Unexpectedly, in the ternary complex with a DNA:DNA duplex and dCTP, the "fingers" subdomain of Polα is in the open conformation. Polα induces conformational changes in the DNA and hybrid duplexes to produce the universal double helix form. Pre-steady-state kinetic studies indicated for both duplex types that chemical catalysis rather than product release is the rate-limiting step. Moreover, human Polα extended DNA primers with higher efficiency but lower processivity than it did with RNA and chimeric primers. Polα has a substantial propensity to make errors during DNA synthesis, and we observed that its fidelity depends on the type of sugar at the primer 3'-end. A detailed structural comparison of Polα with other replicative DNA polymerases disclosed common features and some differences, which may reflect the specialization of each polymerase in genome replication.

Comment on "The [4Fe4S] cluster of human DNA primase functions as a redox switch using DNA charge transport".

O'Brien et al (Research Article, 24 February 2017, eaag1789) proposed a novel mechanism of primase function based on redox activity of the iron-sulfur cluster buried inside the C-terminal domain of the large primase subunit (p58C). Serious problems in the experimental design and data interpretation raise concerns about the validity of the conclusions.

Crystal structure of the human Polϵ B-subunit in complex with the C-terminal domain of the catalytic subunit.

The eukaryotic B-family DNA polymerases include four members: Polα, Polδ, Polϵ, and Polζ, which share common architectural features, such as the exonuclease/polymerase and C-terminal domains (CTDs) of catalytic subunits bound to indispensable B-subunits, which serve as scaffolds that mediate interactions with other components of the replication machinery. Crystal structures for the B-subunits of Polα and Polδ/Polζ have been reported: the former within the primosome and separately with CTD and the latter with the N-terminal domain of the C-subunit. Here we present the crystal structure of the human Polϵ B-subunit (p59) in complex with CTD of the catalytic subunit (p261C). The structure revealed a well defined electron density for p261C and the phosphodiesterase and oligonucleotide/oligosaccharide-binding domains of p59. However, electron density was missing for the p59 N-terminal domain and for the linker connecting it to the phosphodiesterase domain. Similar to Polα, p261C of Polϵ contains a three-helix bundle in the middle and zinc-binding modules on each side. Intersubunit interactions involving 11 hydrogen bonds and numerous hydrophobic contacts account for stable complex formation with a buried surface area of 3094 Å2 Comparative structural analysis of p59-p261C with the corresponding Polα complex revealed significant differences between the B-subunits and CTDs, as well as their interaction interfaces. The B-subunit of Polδ/Polζ also substantially differs from B-subunits of either Polα or Polϵ. This work provides a structural basis to explain biochemical and genetic data on the importance of B-subunit integrity in replisome function in vivo.

Elaborated Action of the Human Primosome.

The human primosome is a 340-kilodalton complex of primase (DNA-dependent RNA polymerase) and DNA polymerase α, which initiates genome replication by synthesizing chimeric RNA-DNA primers for DNA polymerases δ and ϵ. Accumulated biochemical and structural data reveal the complex mechanism of concerted primer synthesis by two catalytic centers. First, primase generates an RNA primer through three steps: initiation, consisting of dinucleotide synthesis from two nucleotide triphosphates; elongation, resulting in dinucleotide extension; and termination, owing to primase inhibition by a mature 9-mer primer. Then Polα, which works equally well on DNA:RNA and DNA:DNA double helices, intramolecularly catches the template primed by a 9mer RNA and extends the primer with dNTPs. All primosome transactions are highly coordinated by autoregulation through the alternating activation/inhibition of the catalytic centers. This coordination is mediated by the small C-terminal domain of the primase accessory subunit, which forms a tight complex with the template:primer, shuttles between the primase and DNA polymerase active sites, and determines their access to the substrate.

Divalent ions attenuate DNA synthesis by human DNA polymerase α by changing the structure of the template/primer or by perturbing the polymerase reaction.

DNA polymerases (pols) are sophisticated protein machines operating in the replication, repair and recombination of genetic material in the complex environment of the cell. DNA pol reactions require at least two divalent metal ions for the phosphodiester bond formation. We explore two understudied roles of metals in pol transactions with emphasis on polα, a crucial enzyme in the initiation of DNA synthesis. We present evidence that the combination of many factors, including the structure of the template/primer, the identity of the metal, the metal turnover in the pol active site, and the influence of the concentration of nucleoside triphosphates, affect DNA pol synthesis. On the poly-dT70 template, the increase of Mg(2+) concentration within the range typically used for pol reactions led to the severe loss of the ability of pol to extend DNA primers and led to a decline in DNA product sizes when extending RNA primers, simulating the effect of "counting" of the number of nucleotides in nascent primers by polα. We suggest that a high Mg(2+) concentration promotes the dynamic formation of unconventional DNA structure(s), thus limiting the apparent processivity of the enzyme. Next, we found that Zn(2+) supported robust polα reactions when the concentration of nucleotides was above the concentration of ions; however, there was only one nucleotide incorporation by the Klenow fragment of DNA pol I. Zn(2+) drastically inhibited polα, but had no effect on Klenow, when Mg(2+) was also present. It is possible that Zn(2+) perturbs metal-mediated transactions in pol active site, for example affecting the step of pyrophosphate removal at the end of each pol cycle necessary for continuation of polymerization.

Mechanism of Concerted RNA-DNA Primer Synthesis by the Human Primosome.

The human primosome, a 340-kilodalton complex of primase and DNA polymerase α (Polα), synthesizes chimeric RNA-DNA primers to be extended by replicative DNA polymerases δ and ϵ. The intricate mechanism of concerted primer synthesis by two catalytic centers was an enigma for over three decades. Here we report the crystal structures of two key complexes, the human primosome and the C-terminal domain of the primase large subunit (p58C) with bound DNA/RNA duplex. These structures, along with analysis of primase/polymerase activities, provide a plausible mechanism for all transactions of the primosome including initiation, elongation, accurate counting of RNA primer length, primer transfer to Polα, and concerted autoregulation of alternate activation/inhibition of the catalytic centers. Our findings reveal a central role of p58C in the coordinated actions of two catalytic domains in the primosome and ultimately could impact the design of anticancer drugs.

Insight into the Human DNA Primase Interaction with Template-Primer.

DNA replication in almost all organisms depends on the activity of DNA primase, a DNA-dependent RNA polymerase that synthesizes short RNA primers of defined size for DNA polymerases. Eukaryotic and archaeal primases are heterodimers consisting of small catalytic and large accessory subunits, both of which are necessary for the activity. The mode of interaction of primase subunits with substrates during the various steps of primer synthesis that results in the counting of primer length is not clear. Here we show that the C-terminal domain of the large subunit (p58C) plays a major role in template-primer binding and also defines the elements of the DNA template and the RNA primer that interact with p58C. The specific mode of interaction with a template-primer involving the terminal 5'-triphosphate of RNA and the 3'-overhang of DNA results in a stable complex between p58C and the DNA/RNA duplex. Our results explain how p58C participates in RNA synthesis and primer length counting and also indicate that the binding site for initiating NTP is located on p58C. These findings provide notable insight into the mechanism of primase function and are applicable for DNA primases from other species.