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...

Autotrophic ammonia-oxidizing bacteria use the essential enzyme ammonia monooxygenase (AMO) to transform ammonia to hydroxylamine. The amo operon consists of at least three genes, amoC, amoA, and amoB; amoA encodes the subunit containing the putative enzyme active site. The use of the amo genes as functional markers for ammonia-oxidizing bacteria in environmental applications requires knowledge of the diversity of the amo operon on several levels: (1) the copy number of the operon in the genome, (2) the arrangement of the three genes in an individual operon, and (3) the primary sequence of the individual genes. We present a database of amo gene sequences for pure cultures of ammonia-oxidizing bacteria representing both the beta- and the gamma-subdivision of Proteobacteria in the following genera: Nitrosospira (6 strains), Nitrosomonas (5 strains) and Nitrosococcus (2 strains). The amo operon was found in multiple (2-3) nearly identical copies in the beta-subdivision representatives but in single copies in the gamma-subdivision ammonia oxidizers. The analysis of the deduced amino acid sequence revealed strong conservation for all three Amo peptides in both primary and secondary structures. For the amoA gene within the beta-subdivision, nucleotide identity values are approximately 85% within the Nitrosomonas or the Nitrosospira groups, but approximately 75% when comparing between these groups. Conserved regions in amoA and amoC were identified and used as primer sites for PCR amplification of amo genes from pure cultures, enrichments and the soil environment. The intergenic region between amoC and amoA is variable in length and may be used to profile the community of ammonia-oxidizing bacteria in environmental samples. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00203-001-0369-z.

A first assessment of anammox activity, which is a unique N2 emission process, was conducted in samples of coastal marine sediment from Japan with a 15 N tracer. The occurrence and diversity of bacteria possibly responsible for the anammox process were also evaluated by selective PCR-amplification of the 16S rRNA gene for known anammox bacteria. Anammox activity, detected by measuring 14 N 15 N gas production, was only found in samples collected at the intertidal sand bank located at the Yodo River estuary. In the Yodo River samples, 16S rRNA gene fragments affiliated with the known anammox bacterial lineage were also recovered, and the two major phylotypes were both "Candidatus Scalindua wagneri" relatives with 95% and 98% sequence similarity. Even from the other samples in which no recognizable anammox activity was detected, 16S rRNA gene fragments related to known anammox bacteria, but not to "Ca. S. wagneri", were detected. This is the first report of anammox-mediated N2 emission in coastal marine environments in Japan. Notably, the PCR-based analysis allowed us to discover unexpected phylogenetic diversity of anammox bacteria-related 16S rRNA gene sequences. The selective PCR primer set developed in this study could be a powerful tool to unveil the ecology of anammox bacteria in natural environments.

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.