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.

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.

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.

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.

Crystal Structure of the Human Pol α B Subunit in Complex with the C-terminal Domain of the Catalytic Subunit.

In eukaryotic DNA replication, short RNA-DNA hybrid primers synthesized by primase-DNA polymerase α (Prim-Pol α) are needed to start DNA replication by the replicative DNA polymerases, Pol δ and Pol ϵ. The C terminus of the Pol α catalytic subunit (p180C) in complex with the B subunit (p70) regulates the RNA priming and DNA polymerizing activities of Prim-Pol α. It tethers Pol α and primase, facilitating RNA primer handover from primase to Pol α. To understand these regulatory mechanisms and to reveal the details of human Pol α organization, we determined the crystal structure of p70 in complex with p180C. The structured portion of p70 includes a phosphodiesterase (PDE) domain and an oligonucleotide/oligosaccharide binding (OB) domain. The N-terminal domain and the linker connecting it to the PDE domain are disordered in the reported crystal structure. The p180C adopts an elongated asymmetric saddle shape, with a three-helix bundle in the middle and zinc-binding modules (Zn1 and Zn2) on each side. The extensive p180C-p70 interactions involve 20 hydrogen bonds and a number of hydrophobic interactions resulting in an extended buried surface of 4080 Å(2). Importantly, in the structure of the p180C-p70 complex with full-length p70, the residues from the N-terminal to the OB domain contribute to interactions with p180C. The comparative structural analysis revealed both the conserved features and the differences between the human and yeast Pol α complexes.

Crystal structure of the human primase.

DNA replication in bacteria and eukaryotes requires the activity of DNA primase, a DNA-dependent RNA polymerase that lays short RNA primers for DNA polymerases. Eukaryotic and archaeal primases are heterodimers consisting of small catalytic and large accessory subunits, both of which are necessary for RNA primer synthesis. Understanding of RNA synthesis priming in eukaryotes is currently limited due to the lack of crystal structures of the full-length primase and its complexes with substrates in initiation and elongation states. Here we report the crystal structure of the full-length human primase, revealing the precise overall organization of the enzyme, the relative positions of its functional domains, and the mode of its interaction with modeled DNA and RNA. The structure indicates that the dramatic conformational changes in primase are necessary to accomplish the initiation and then elongation of RNA synthesis. The presence of a long linker between the N- and C-terminal domains of p58 provides the structural basis for the bulk of enzyme's conformational flexibility. Deletion of most of this linker affected the initiation and elongation steps of the primer synthesis.

Structural basis for inhibition of DNA replication by aphidicolin.

Natural tetracyclic diterpenoid aphidicolin is a potent and specific inhibitor of B-family DNA polymerases, haltering replication and possessing a strong antimitotic activity in human cancer cell lines. Clinical trials revealed limitations of aphidicolin as an antitumor drug because of its low solubility and fast clearance from human plasma. The absence of structural information hampered the improvement of aphidicolin-like inhibitors: more than 50 modifications have been generated so far, but all have lost the inhibitory and antitumor properties. Here we report the crystal structure of the catalytic core of human DNA polymerase α (Pol α) in the ternary complex with an RNA-primed DNA template and aphidicolin. The inhibitor blocks binding of dCTP by docking at the Pol α active site and by rotating the template guanine. The structure provides a plausible mechanism for the selectivity of aphidicolin incorporation opposite template guanine and explains why previous modifications of aphidicolin failed to improve its affinity for Pol α. With new structural information, aphidicolin becomes an attractive lead compound for the design of novel derivatives with enhanced inhibitory properties for B-family DNA polymerases.

Crystal structure of HIV-1 Tat complexed with human P-TEFb and AFF4.

Developing anti-viral therapies targeting HIV-1 transcription has been hampered by the limited structural knowledge of the proteins involved. HIV-1 hijacks the cellular machinery that controls RNA polymerase II elongation through an interaction of HIV-1 Tat with the positive transcription elongation factor P-TEFb, which interacts with an AF4 family member (AFF1/2/3/4) in the super elongation complex (SEC). Because inclusion of Tat•P-TEFb into the SEC is critical for HIV transcription, we have determined the crystal structure of the Tat•AFF4•P-TEFb complex containing HIV-1 Tat (residues 1-48), human Cyclin T1 (1-266), human Cdk9 (7-332), and human AFF4 (27-69). Tat binding to AFF4•P-TEFb causes concerted structural changes in AFF4 via a shift of helix H5' of Cyclin T1 and the α-3 10 helix of AFF4. The interaction between Tat and AFF4 provides structural constraints that explain tolerated Tat mutations. Analysis of the Tat-binding surface of AFF4 coupled with modeling of all other AF4 family members suggests that AFF1 and AFF4 would be preferred over AFF2 or AFF3 for interaction with Tat•P-TEFb. The structure establishes that the Tat-TAR recognition motif (TRM) in Cyclin T1 interacts with both Tat and AFF4, leading to the exposure of arginine side chains for binding to TAR RNA. Furthermore, modeling of Tat Lys28 acetylation suggests that the acetyl group would be in a favorable position for H-bond formation with Asn257 of TRM, thereby stabilizing the TRM in Cyclin T1, and provides a structural basis for the modulation of TAR RNA binding by acetylation of Tat Lys28.

Crystallization and preliminary X-ray diffraction analysis of human DNA primase.

Human primase synthesizes RNA primers and transfers them to the active site of Pol α with subsequent extension with dNTPs. Human primase is a heterodimer of two subunits: a small catalytic subunit (p49) and a large subunit (p58). The structural details of the initiation and elongation steps of primer synthesis, as well as primer length counting, are not known. To address these questions, structural studies of human primase were initiated. Two types of crystals were obtained. The best diffracting crystals belonged to space group P1, with unit-cell parameters a = 86.2, b = 88.9, c = 94.68 Å, α = 93.82, ß = 96.57, γ = 111.72°, and contained two heterodimers of full-length p49 and p59 subunits in the asymmetric unit.

Structural basis of Ets1 cooperative binding to widely separated sites on promoter DNA.

Ets1 is a member of the Ets family of transcription factors. Ets1 is expressed in autoinhibited form and its DNA binding depends on partner proteins bound to adjacent sequences or the relative positioning of a second Ets-binding site (EBS). The autoinhibition of Ets1 is mediated by structural coupling of regions flanking the DNA-binding domain. The NMR structure of Ets1 revealed that the inhibitory regions comprised of helices HI1 and HI2 and H4 are packed together on the Ets domain to form an inhibitory module. The crystal structure of Ets1 unexpectedly revealed a homodimer in which homodimerisation occurs via swapping of HI1 helices. Modeling of DNA binding indicates that the Ets1 dimer can bind to two antiparallel pieces of DNA. To verify this, we crystallized and solved the structure of the complex comprised of Ets1 dimer and two pieces of DNA. DNA binding by Ets1 dimer resulted in formation of additional intermolecular protein•DNA interactions, implying that the complex formation is cooperative.

Crystal structure of the C-terminal domain of human DNA primase large subunit: implications for the mechanism of the primase-polymerase α switch.

DNA polymerases cannot synthesize DNA without a primer, and DNA primase is the only specialized enzyme capable of de novo synthesis of short RNA primers. In eukaryotes, primase functions within a heterotetrameric complex in concert with a tightly bound DNA polymerase α (Pol α). In humans, the Pol α part is comprised of a catalytic subunit (p180) and an accessory subunit B (p70), and the primase part consists of a small catalytic subunit (p49) and a large essential subunit (p58). The latter subunit participates in primer synthesis, counts the number of nucleotides in a primer, assists the release of the primer-template from primase and transfers it to the Pol α active site. Recently reported crystal structures of the C-terminal domains of the yeast and human enzymes' large subunits provided critical information related to their structure, possible sites for binding of nucleotides and template DNA, as well as the overall organization of eukaryotic primases. However, the structures also revealed a difference in the folding of their proposed DNA-binding fragments, raising the possibility that yeast and human proteins are functionally different. Here we report new structure of the C-terminal domain of the human primase p58 subunit. This structure exhibits a fold similar to a fold reported for the yeast protein but different than a fold reported for the human protein. Based on a comparative analysis of all three C-terminal domain structures, we propose a mechanism of RNA primer length counting and dissociation of the primer-template from primase by a switch in conformation of the ssDNA-binding region of p58.

Structural basis of Ets1 cooperative binding to palindromic sequences on stromelysin-1 promoter DNA.

Ets1 is a member of the Ets family of transcription factors. Ets1 is autoinhibited and its activation requires heterodimerization with a partner protein or DNA-mediated homodimerization for cooperative DNA binding. In the latter case, Ets1 molecules bind to palindromic sequences in which two Ets-binding sites (EBS) are separated by four base pairs, for example in the promoters of stromelysin-1 and p53. Interestingly, counteraction of autoinhibition requires the autoinhibitory region encoded by exon VII of the gene. The structural basis for the requirement of autoinhibitory sequences for Ets1 binding to palindromic EBS still remains unresolved. Here we report the crystal structure of two Ets1 molecules bound to an EBS palindrome of the stromelysin-1 promoter DNA, providing a plausible explanation for the requirement of exon VII-encoded sequences for Ets1 cooperative DNA binding. The proposed mechanism was verified both in vitro by surface plasmon resonance and in vivo by transcription-based assays.