To characterize proteins associated with active transcription complexes, we purified RNA polymerase II (pol II) from Saccharomyces cerevisiae after fixing live cells with formaldehyde. The approach mimics ChIP and requires solubilizing cross-linked complexes with sonication. Pol II was affinity-purified, and associated proteins were identified by MS. Several classes of proteins depended on cros...

Electron cryomicroscopy reconstructions of elongating RNA polymerase (Pol) III at 3.9 Å resolution and of unbound Pol III (apo Pol III) in two distinct conformations at 4.6 Å and 4.7 Å resolution allow the construction of complete atomic models of Pol III and provide new functional insights into the adaption of Pol III to fulfill its specific transcription tasks.

The macromolecular assembly required to initiate transcription of protein-coding genes, known as the Pre-Initiation Complex (PIC), consists of multiple protein complexes and is approximately 3.5 MDa in size. At the heart of this assembly is the Mediator complex, which helps regulate PIC activity and interacts with the RNA polymerase II (pol II) enzyme. The structure of the human Mediator-pol II...

The papillomavirus (PV) helicase protein E1 recruits components of the cellular DNA replication machinery to the PV replication fork, such as Replication Protein A (RPA), DNA polymerase α-primase (pol α) and topoisomerase I (topo I). Here we show that E1 binds to DNA polymerase ϵ (pol ϵ) and dramatically stimulates the DNA synthesis activity of pol ϵ. This stimulation of pol ϵ by E1 is highly s...

The accessory protein polymerase (pol) gammaB of the human mitochondrial DNA polymerase stimulates the synthetic activity of the catalytic subunit. pol gammaB functions by both accelerating the polymerization rate and enhancing polymerase-DNA interaction, thereby distinguishing itself from the accessory subunits of other DNA polymerases. The molecular basis for the unique functions of human pol...

All cells contain specialized translesion DNA polymerases that replicate past sites of DNA damage. We find that Escherichia coli translesion DNA polymerase II (Pol II) and polymerase IV (Pol IV) function with DnaB helicase and regulate its rate of unwinding, slowing it to as little as 1 bp/s. Furthermore, Pol II and Pol IV freely exchange with the polymerase III (Pol III) replicase on the beta-...

RNA polymerase (Pol) II transcribes protein-coding genes in the nucleus of eukaryotic cells and consists of 12 polypeptide subunits. It is unknown how Pol II is imported into the nucleus. Here we show that Pol II nuclear import requires the protein Iwr1 and provide evidence for cyclic Iwr1 function. Iwr1 binds Pol II in the active center cleft between the two largest subunits, maybe facilitatin...

DNA polymerase beta (Pol beta) is important for the base excision repair (BER) pathway. Overexpression of Pol beta is frequently found in cancer cells and is thought to be associated with tumorigenesis. In this study, we examined BER fidelity in extracts derived from a human lymphoblastoid cell line that over expresses Pol beta compared to normal control cells. Using an in vitro mutagenesis ass...

BACKGROUND Human RNA polymerase III (pol III) transcription is regulated by several factors, including the tumor suppressors P53 and Rb, and the proto-oncogene c-Myc. In yeast, which lacks these proteins, a central regulator of pol III transcription, called Maf1, has been described. Maf1 is required for repression of pol III transcription in response to several signal transduction pathways and ...

In Archaea repair of uracil and hypoxanthine, which arise by deamination of cytosine and adenine, respectively, is initiated by three enzymes: Uracil-DNA-glycosylase (UDG, which recognises uracil); Endonuclease V (EndoV, which recognises hypoxanthine); and Endonuclease Q (EndoQ), (which recognises both uracil and hypoxanthine). Two archaeal DNA polymerases, Pol-B and Pol-D, are inhibited by dea...