Residues located in the primase domain of the bacteriophage T7 primase-helicase are essential for loading the hexameric complex onto DNA.

The T7 primase-helicase plays a pivotal role in the replication of T7 DNA. Using affinity isolation of peptide-nucleic acid crosslinks and mass spectrometry, we identify protein regions in the primase-helicase and T7 DNA polymerase that form contacts with the RNA primer and DNA template. The contacts between nucleic acids and the primase domain of the primase-helicase are centered in the RNA polymerase subdomain of the primase domain, in a cleft between the N-terminal subdomain and the topoisomerase-primase fold. We demonstrate that residues along a beta sheet in the N-terminal subdomain that contacts the RNA primer are essential for phage growth and primase activity in vitro. Surprisingly, we found mutations in the primase domain that had a dramatic effect on the helicase. Substitution of a residue conserved in other DnaG-like enzymes, R84A, abrogates both primase and helicase enzymatic activities of the T7 primase-helicase. Alterations in this residue also decrease binding of the primase-helicase to ssDNA. However, mass photometry measurements show that these mutations do not interfere with the ability of the protein to form the active hexamer.

Measuring the Complex Effects of the Single-Stranded DNA-Binding Protein gp2.5 on Primer Synthesis and Extension by the Bacteriophage T7 Replisome.

The single-stranded DNA-binding protein gp2.5 of bacteriophage T7 plays myriad functions in the replication of phage genomes. In addition to interacting with ssDNA, gp2.5 binds to the T7 DNA polymerase and primase/helicase proteins, regulating their enzymatic activities. Here we describe in vitro methods to examine the effects of gp2.5 on primer synthesis and extension by the T7 replisome.

Catalytically inactive T7 DNA polymerase imposes a lethal replication roadblock.

Bacteriophage T7 encodes its own DNA polymerase, the product of gene 5 (gp5). In isolation, gp5 is a DNA polymerase of low processivity. However, gp5 becomes highly processive upon formation of a complex with Escherichia coli thioredoxin, the product of the trxA gene. Expression of a gp5 variant in which aspartate residues in the metal-binding site of the polymerase domain were replaced by alanine is highly toxic to E. coli cells. This toxicity depends on the presence of a functional E. coli trxA allele and T7 RNA polymerase-driven expression but is independent of the exonuclease activity of gp5. In vitro, the purified gp5 variant is devoid of any detectable polymerase activity and inhibited DNA synthesis by the replisomes of E. coli and T7 in the presence of thioredoxin by forming a stable complex with DNA that prevents replication. On the other hand, the highly homologous Klenow fragment of DNA polymerase I containing an engineered gp5 thioredoxin-binding domain did not exhibit toxicity. We conclude that gp5 alleles encoding inactive polymerases, in combination with thioredoxin, could be useful as a shutoff mechanism in the design of a bacterial cell-growth system.

B2 and ALU retrotransposons are self-cleaving ribozymes whose activity is enhanced by EZH2.

Transposable elements make up half of the mammalian genome. One of the most abundant is the short interspersed nuclear element (SINE). Among their million copies, B2 accounts for ∼350,000 in the mouse genome and has garnered special interest because of emerging roles in epigenetic regulation. Our recent work demonstrated that B2 RNA binds stress genes to retard transcription elongation. Although epigenetically silenced, B2s become massively up-regulated during thermal and other types of stress. Specifically, an interaction between B2 RNA and the Polycomb protein, EZH2, results in cleavage of B2 RNA, release of B2 RNA from chromatin, and activation of thermal stress genes. Although an established RNA-binding protein and histone methyltransferase, EZH2 is not known to be a nuclease. Here, we provide evidence for the surprising conclusion that B2 is a self-cleaving ribozyme. Ribozyme activity depends on Mg+2 and monovalent cations but is resistant to protease treatment. However, contact with EZH2 accelerates cleavage rate by >100-fold, suggesting that EZH2 promotes a cleavage-competent RNA conformation. B2 modification-interference analysis demonstrates that phosphorothioate changes at A and C nucleotides can substitute for EZH2. B2 nucleotides 45 to 55 and 100 to 101 are essential for activity. Finally, another family of SINEs, the human ALU element, also produces a self-cleaving RNA and is cleaved during T-cell activation as well as thermal and endoplasmic reticulum (ER) stress. Thus, B2/ALU SINEs may be classified as "epigenetic ribozymes" that function as transcriptional switches during stress. Given their high copy numbers, B2 and ALU may represent the predominant ribozyme activity in mammalian cells.

Gp2.5, the multifunctional bacteriophage T7 single-stranded DNA binding protein.

The essential bacteriophage T7-encoded single-stranded DNA binding protein is the nexus of T7 DNA metabolism. Multiple layers of macromolecular interactions mediate its function in replication, recombination, repair, and the maturation of viral genomes. In addition to binding ssDNA, the protein binds to DNA polymerase and DNA helicase, regulating their activities. The protein displays potent homologous DNA annealing activity, underscoring its role in recombination.

Deep-sea vent phage DNA polymerase specifically initiates DNA synthesis in the absence of primers.

A DNA polymerase is encoded by the deep-sea vent phage NrS-1. NrS-1 has a unique genome organization containing genes that are predicted to encode a helicase and a single-stranded DNA (ssDNA)-binding protein. The gene for an unknown protein shares weak homology with the bifunctional primase-polymerases (prim-pols) from archaeal plasmids but is missing the zinc-binding domain typically found in primases. We show that this gene product has efficient DNA polymerase activity and is processive in DNA synthesis in the presence of the NrS-1 helicase and ssDNA-binding protein. Remarkably, this NrS-1 DNA polymerase initiates DNA synthesis from a specific template DNA sequence in the absence of any primer. The de novo DNA polymerase activity resides in the N-terminal domain of the protein, whereas the C-terminal domain enhances DNA binding.

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins.

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 replisome is one of the simplest replication systems known, composed of only four proteins, which is an attractive feature for biochemical experiments. Special emphasis is placed on the synthesis of ribonucleotide primers by the T7 primase-helicase, which are used by DNA polymerase to initiate DNA synthesis. Because the mechanisms of DNA replication are conserved across evolution, these protocols should be applicable, or useful as a conceptual springboard, to investigators using other model systems. The protocols described here are highly sensitive and an experienced investigator can perform these experiments and obtain data for analysis in about a day. The only specialized piece of equipment required is a rapid-quench flow instrument, but this piece of equipment is relatively common and available from various commercial sources. The major drawbacks of these assays, however, include the use of radioactivity and the relative low throughput.

Destabilization of B2 RNA by EZH2 Activates the Stress Response.

More than 98% of the mammalian genome is noncoding, and interspersed transposable elements account for ∼50% of noncoding space. Here, we demonstrate that a specific interaction between the polycomb protein EZH2 and RNA made from B2 SINE retrotransposons controls stress-responsive genes in mouse cells. In the heat-shock model, B2 RNA binds stress genes and suppresses their transcription. Upon stress, EZH2 is recruited and triggers cleavage of B2 RNA. B2 degradation in turn upregulates stress genes. Evidence indicates that B2 RNA operates as a "speed bump" against advancement of RNA polymerase II, and temperature stress releases the brakes on transcriptional elongation. These data attribute a new function to EZH2 that is independent of its histone methyltransferase activity and reconcile how EZH2 can be associated with both gene repression and activation. Our study reveals that EZH2 and B2 together control activation of a large network of genes involved in thermal stress.

Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome.

DNA replication occurs semidiscontinuously due to the antiparallel DNA strands and polarity of enzymatic DNA synthesis. Although the leading strand is synthesized continuously, the lagging strand is synthesized in small segments designated Okazaki fragments. Lagging-strand synthesis is a complex event requiring repeated cycles of RNA primer synthesis, transfer to the lagging-strand polymerase, and extension effected by cooperation between DNA primase and the lagging-strand polymerase. We examined events controlling Okazaki fragment initiation using the bacteriophage T7 replication system. Primer utilization by T7 DNA polymerase is slower than primer formation. Slow primer release from DNA primase allows the polymerase to engage the complex and is followed by a slow primer handoff step. The T7 single-stranded DNA binding protein increases primer formation and extension efficiency but promotes limited rounds of primer extension. We present a model describing Okazaki fragment initiation, the regulation of fragment length, and their implications for coordinated leading- and lagging-strand DNA synthesis.

Regulatory interactions between RNA and polycomb repressive complex 2.

Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that is localized to thousands of mammalian genes. Though important to human disease and as a drug target, how PRC2 is recruited remains unclear. One model invokes cis-regulatory RNA. Herein, we biochemically and functionally probe PRC2's recognition of RNA using the X-inactivation model. We observe surprisingly high discriminatory capabilities. While SUZ12 and JARID2 subunits can bind RNA, EZH2 has highest affinity and is somewhat promiscuous. EED regulates the affinity of EZH2 for RNA, lending greater specificity to PRC2-RNA interactions. Intriguingly, while RNA is crucial for targeting, RNA inhibits EZH2's catalytic activity. JARID2 weakens PRC2's binding to RNA and relieves catalytic inhibition. We propose that RNA guides PRC2 to its target but inhibits its enzymatic activity until PRC2 associates with JARID2 on chromatin. Our study provides a molecular view of regulatory interactions between RNA and PRC2 at the chromatin interface.

EUS-guided pancreaticogastrostomy: analysis of its efficacy to drain inaccessible pancreatic ducts.

BACKGROUND: EUS-guided pancreaticogastrostomy (EPG) has been reported as an alternative to surgery in cases of pancreatic stricture where ERCP is unsuccessful. OBJECTIVE: We analyzed our 3-year experience with this innovative technique. DESIGN: Patients with failed ERCP for pancreatic drainage were offered EPG over a 3-year period and were followed up prospectively in terms of clinical and radiologic response. SETTING: Tertiary care center offering ERCP and interventional EUS. PATIENTS: Thirteen patients were included in this study. Seven had surgical diversion Six patients had unaltered enteral anatomy and stricture related to chronic pancreatitis (3), gallstone pancreatitis (2), and intraductal pancreatic mucinous neoplasm (1). INTERVENTION: EUS-guided puncture and opacification of the pancreatic duct was performed, creating a transgastric fistula with placement of a guidewire into the main pancreatic duct and subsequent ductal decompression with a plastic endoprosthesis. MAIN OUTCOME MEASUREMENTS: Mean main pancreatic duct size, pain score, and weight before and after intervention. RESULTS: Ten patients had successful endoprosthesis placement across the pancreaticogastric fistula. One patient underwent brush cytologic study, which diagnosed pancreatic malignancy, and underwent surgical resection. After a mean follow-up of 14 months, the mean pancreatic duct size in treated patients decreased from 4.6 to 3.0 mm (P = .01); the pain score decreased from 7.3 to 3.6 (P = .01). Complications included one case of bleeding requiring hemoclip placement and 1 case of contained perforation. LIMITATIONS: Pilot study from a single center. CONCLUSIONS: EPG is a safe and feasible alternative to surgical intervention in this subgroup of patients where conventional ERCP is not possible.

Interventional EUS-guided cholangiography: evaluation of a technique in evolution.

BACKGROUND: Interventional EUS-guided cholangiography (IEUC) has been increasingly used as an alternative to percutaneous transhepatic cholangiography (PTC) in cases of biliary obstruction when ERCP is unsuccessful. OBJECTIVE: We reviewed our experience and technique used for this procedure. DESIGN: Over a 3-year period, ending July 2005, patients with a failed ERCP were offered an IEUC. SETTING: Tertiary care center offering ERCP and interventional EUS. PATIENTS: Twenty-eight patients were candidates for IEUC. Two patients had bleeding masses and were referred to interventional radiology, 1 patient had a large mass occupying the duodenal lumen, and 2 patients refused IEUC. INTERVENTION: EUS was used to access the biliary system after which a guidewire was advanced antegrade across the obstruction. Either rendezvous with retrograde or antegrade drainage was then accomplished. MAIN OUTCOME MEASUREMENTS: Efficacy and safety of IEUC for biliary decompression. RESULTS: IEUC was successfully performed in 23 patients, with a transgastric-transhepatic (intrahepatic) approach in 13 cases and transenteric-transcholedochal (extrahepatic) approach in 10 cases. Therapeutic benefit was achieved in 21 patients: 18 underwent successful stent deployment across the stricture, whereas 3 patients required a choledochoenteric fistula formation. Complications included 1 case of bile leak, 2 cases of self-limited pneumoperitoneum, and 1 case of minor bleeding. LIMITATIONS: Single-center experience of 2 operators. CONCLUSIONS: IEUC appears efficacious in patients in whom ERCP is unsuccessful and is evolving as an attractive alternative to PTC. Intrahepatic access to the biliary system appears safer than the extrahepatic approach.