Regulation of the expression of the human immunodeficiency virus (HIV) genome is accomplished in large part by controlling transcription elongation. The viral protein Tat hijacks the host cell's RNA polymerase II elongation control machinery through interaction with the positive transcription elongation factor, P-TEFb, and directs the factor to promote productive elongation of HIV mRNA. Here we describe the crystal structure of the Tat•P-TEFb complex containing HIV-1 Tat, human Cdk9, and human Cyclin T1. Tat adopts a structure complementary to the surface of P-TEFb and makes extensive contacts, mainly with the Cyclin T1 subunit of P-TEFb, but also with the T-loop of the Cdk9 subunit. The structure provides a plausible explanation for the tolerance of Tat to sequence variations at certain sites. Importantly, Tat induces significant conformational changes in P-TEFb. This finding lays a foundation for the design of compounds that would specifically inhibit the Tat•P-TEFb complex and block HIV replication.

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...

The eukaryotic DNA polymerase δ (Pol δ) participates in genome replication, homologous recombination, DNA repair and damage tolerance. Regulation of the plethora of Pol δ functions depends on the interaction between the second (p50) and third (p66) non-catalytic subunits. We report the crystal structure of p50•p66 N complex featuring oligonucleotide binding and phosphodiesterase domains in p50 and winged helix-turn-helix N-terminal domain in p66. Disruption of the interaction between the yeast orthologs of p50 and p66 by strategic amino acid changes leads to cold-sensitivity, sensitivity to hydroxyurea and to reduced UV mutagenesis, mimicking the phenotypes of strains where the third subunit of Pol δ is absent. The second subunits of all B family replicative DNA polymerases in archaea and eukaryotes, except Pol δ, share a three-domain structure similar to p50•p66 N , raising the possibility that a portion of the gene encoding p66 was derived from the second subunit gene relatively late in evolution.

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...