kinetics-lagging-strand-dna-synthesis-vitro-bacteriophage-t7

Kinetics of Lagging-strand DNA Synthesis <em>In Vitro</em> 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 ...

Harvard Medical School

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.

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

EUS-guided pancreaticogastrostomy (EPG) has been reported as an alternative to surgery in cases of pancreatic stricture where ERCP is unsuccessful.

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

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.

Regulatory interactions between RNA and polycomb repressive complex 2.

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.

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

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.

Alfredo J. Hernandez

Department of Biological Chemistry and Molecular Pharmacology

Harvard Medical School

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

Alfredo J. Hernandez

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biological Chemistry and Molecular Pharmacology

Harvard Medical School

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

Department of Biological Chemistry and Molecular Pharmacology