The human primosome, a 340-kilodalton complex of primase and DNA polymerase ␣ (Pol␣), synthesizes chimeric RNA-DNA primers to be extended by replicative DNA polymerases ␦ and ⑀. 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 (p58 C ) 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 Pol␣, and concerted autoregulation of alternate activation/inhibition of the catalytic centers. Our findings reveal a central role of p58 C in the coordinated actions of two catalytic domains in the primosome and ultimately could impact the design of anticancer drugs.In eukaryotes, the primosome, a tight complex of DNA primase and DNA polymerase ␣ (Pol␣), 4 synthesizes primers for both leading and lagging strands in a highly coordinated fashion (1, 2). The primosome is indispensable for initiation of replication and has a large impact on genome stability (3-6). RNA primer synthesis by primase involves three steps: initiation, elongation, and termination (7,8). During the rate-limiting initiation step, primase binds the DNA template and two ribonucleotide triphosphates (NTPs) and catalyzes the formation of a dinucleotide (9, 10). Further synthesis of the RNA primer is much faster but restricted, because of the intrinsic property of primases to count the primer length and terminate synthesis after incorporation of 8 -10 nucleotides (7). Next, the mature so-called "unit length" RNA primer is intramolecularly translocated to Pol␣ for the subsequent extension by dNTPs, and the primase became inhibited by an unknown mechanism (9,11,12). Orchestration of all these steps requires changes in primosome conformation (13).Human Pol␣ (Fig. 1A) is comprised of a large catalytic subunit (p180) and a smaller accessory subunit (p70), connected by the C-terminal domain of p180 (p180 C ) containing two conserved zinc-binding modules, Zn1 and Zn2 (14 -16). p70 consists of an N-terminal (p70 N ), a phosphodiesterase, and oligonucleotide/oligosaccharide-binding (OB) domains (14, 17). The globular p70 N is attached to the phosphodiesterase via a flexible linker (amino acid residues 79 -156) (14, 18) and participates in interactions with other DNA replication proteins (19). The catalytic core of p180 (p180core) and p180 C -p70 are connected by a 15-residue linker (1251-1265) (13). Human primase consists of catalytic (p49) and regulatory (p58) subunits (20). p58 has two distinct domains, N-terminal (p58 N ) and C-terminal (p58 C ), connected with an 18-residue linker (253-270) (21). p58 N interacts with p49 and connects primase with Pol␣ (22, 23), and an iron-sulfur cluster containing p58 C plays an important role in substrate binding and primase acti...

Background: DNA polymerase (Pol) ␦ is involved in UV light-induced mutagenesis by an unknown mechanism. Results: The C terminus of DNA Pol interacts with accessory subunits of DNA Pol ␦, which is required for UV light-induced mutagenesis. Conclusion: When replication is stalled, accessory subunits of DNA Pol ␦ participate in recruitment of translesion DNA Pol . Significance: This finding provides a novel mechanism of DNA lesion bypass in eukaryotes.

Background: DNA primase synthesizes RNA primers and is indispensable for genome replication.Results: We present a crystal structure of the intact human primase at 2.65 Å resolution. Conclusion:The long linker between two domains of the large subunit is important for RNA priming. Significance: The obtained data provide notable insight into the mechanism of primase function.

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 (p58 C ) plays a major role in template-primer binding and also defines the elements of the DNA template and the RNA primer that interact with p58 C . 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 p58 C and the DNA/RNA duplex. Our results explain how p58 C participates in RNA synthesis and primer length counting and also indicate that the binding site for initiating NTP is located on p58 C . These findings provide notable insight into the mechanism of primase function and are applicable for DNA primases from other species.The four-subunit primase-polymerase ␣ (Prim-Pol␣) 3 complex possessing DNA primase and DNA polymerase active sites is important for genome replication in eukaryotes (1, 2). PrimPol␣ synthesizes the chimeric RNA-DNA primers for replicative DNA polymerases ⑀ and ␦ (3, 4). In humans, the primase heterodimer contains a small catalytic subunit (p49; also known as p48, PRIM1, Pri1, and PriS) and a large regulatory subunit (p58; also known as PRIM2, Pri2, and PriL). Pol␣ is composed of a large catalytic subunit (p180) and a small accessory subunit (p70). p58 and p70 are connected with p180 through the interaction with a small C-terminal domain (p180 C ) that defines the tight coordination of the RNA-and DNA-polymerizing activities (5-7).Eukaryotic primases have a minimal specific recognition site on DNA and only require a pyrimidine to template the 5Ј-terminal nucleotide of the primer (8, 9). RNA primer synthesis begins with a rate-limiting initiation step that includes binding of the DNA template and two NTPs followed by dinucleotide synthesis (10). Subsequently, primase elongates the generated dinucleotide to the unit-length primer (8 -10-mer) and terminates synthesis. This unique counting ability of DNA primases, which results in RNA primers that are optimal for extension by Pol␣, has a complex mechanism that is currently unclear. Recent structural data revealed that the primase active site located on p49 uses the common mechanism of nucleic acids synthesis, where the catalytic aspartates coordinate two divalent ions and the triphosphate moiety of the incoming NTP (11,12).The large subunit of human primase is composed of two separate domains connected with a long linker, which indicates a significant conformational flexibility of this subunit (13). The N-terminal domain (p58 N ) provides a platform for interactions with p49 and P...

Large scale heterogeneous distribution of biodiversity has become a hot topic for ecologists and conservationists. A threat status assessment combined with geographic distribution patterns of threatened plants in China has been conducted at a national scale in this study based upon a distribution database that refers to both specimen records and published references. Currently, 302 threatened plant species are cataloged in the ''National Protected Key Wild Plants'' in China belonging to 92 families and 194 genera. Results of the assessment according to the Categories and Criteria system of The World Conservation Union (IUCN) Red List indicate that three species have been assessed as Extinct in Wild (EW) while a further 79, 99 and 112 species have been assessed as Critically Endangered (CR), Endangered (EN), and Vulnerable (VU), respectively. Distribution patterns of threatened plants were analyzed with GIS to identify areas of high species diversity. It was found that threatened plant species occur unevenly within counties and are concentrated in the following eight hotspots: the central and southern Hengduanshan mountain area; the southeast regions of Yunnan as well as Xishuangbanna and southwestern Guangxi; the southern Hainan island; the border mountainous regions of Guizhou, Hunan and Guangxi provinces; the mountainous regions of southwestern Hubei and northern Hunan; southwestern Zhejiang and western Fujian; central Sichuan and southern Gansu; and the western mountains of Guangdong. Moreover, the 12 counties with the greatest number of threatened plant species represent cumulatively more than 50% of the total listed species and, therefore, are the regions in China that should be prioritized for conservation efforts. By overlapping the map of threatened plant species with the distribution of national nature reserves, a gap was identified in protected areas. This research will ultimately provide insights for prioritizing biodiversity conservation as well as processing the mechanisms of distribution patterns.

Background: DNA polymerase ␣-primase synthesizes chimeric RNA/DNA primers for replicative polymerases. Results: We defined elements that modulate pol␣ and prim activities. Conclusion: The C-terminal domain of the catalytic subunit of polymerase ␣ and the B-subunit regulate the priming of DNA replication. Significance: We provide new information on the regulation of RNA/DNA synthesizing complex that is indispensable for replication in eukaryotes.

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.

The data suggest that higher levels of serum carotenoids, in particular zeaxanthin and lycopene, are associated with a lower likelihood of having exudative AMD. Serum levels of carotenoids were relatively higher in this Chinese cohort than in samples of other ethnicities in previous reports.