Coupling nucleic acid processing enzymes to nanoscale pores allows controlled movement of individual DNA or RNA strands that is reported as an ionic current time series. Hundreds of individual enzyme complexes can be examined in single-file order at high bandwidth and spatial resolution. The bacteriophage phi29 DNA polymerase (phi29 DNAP) is an attractive candidate for this technology, due to its remarkable processivity and high affinity for DNA substrates. Here we show that phi29 DNAP-DNA complexes are stable when captured in an electric field across the α-hemolysin nanopore. DNA substrates were activated for replication at the nanopore orifice by exploiting the 3′-5′ exonuclease activity of wild-type phi29 DNAP to excise a 3′-H terminal residue, yielding a primer strand 3′-OH. In the presence of deoxynucleoside triphosphates, DNA synthesis was initiated, allowing real time detection of numerous sequential nucleotide additions that was limited only by DNA template length. Translocation of phi29 DNAP along DNA substrates was observed in real time at Angstrom scale precision as the template strand was drawn through the nanopore lumen during replication.Single molecule techniques are now used routinely to study nucleic acids in basic science 1 -3 and technology 4 ,5 . Methods using nanoscale pores (nanopores) are advantageous because they can report the length, structure and composition of unmodified DNA or RNA molecules that are captured in single file order 6-9. Data are typically reported as a time series of ionic current as each DNA strand is driven by an applied electric field across a single pore controlled by a voltage-clamped amplifier. Hundreds to thousands of molecules can be examined at high bandwidth and spatial resolution.Recently, the properties of DNA or RNA molecules bound to nucleic acid processing enzymes have been analyzed at a nanopore orifice. The complexes studied include those of single-stranded DNA with Escherichia coli Exonuclease I 10 , RNA with the bacteriophage phi8 ATPase, 11 , and primer/template DNA substrates bound to the 3′-5′-exonuclease deficient versions of two A-family DNA polymerases, the Klenow fragment of E. coli DNA polymerase (KF(exo-)) and bacteriophage T7 DNA polymerase (T7DNAP(exo-)) 12 -16. We have demonstrated that T7DNAP(exo-) could replicate and advance a DNA template held in the α-hemolysin (α-HL) nanopore against an 80 mV applied potential 17. However, due to the low stability of the T7DNAP(exo-)-DNA complex under load, diminished signal * corresponding author: makeson@soe.ucsc.edu. Figure S1 (sequences of 5′-6-FAM, 3′-OH and 5′-6-FAM, 3′-H DNA oligonucleotide substrates used in gel assays); Figure S2 (unbound DNA at 70 mV applied potential); Figure S3 (primer extension gel assays supporting phi2 DNAP-DNA-dGTP ternary complex formation); Figure S4 (amplitude steps in the terminal cascade vary as a function of initial DNA substrate abasic configuration); Supporting Information Available:

The chromatin architecture in promoters is thought to regulate gene expression, but it remains uncertain how most transcription factors (TFs) impact nucleosome position. The MuvB TF complex regulates cell-cycle dependent gene-expression and is critical for differentiation and proliferation during development and cancer. MuvB can both positively and negatively regulate expression, but the structure of MuvB and its biochemical function are poorly understood. Here we determine the overall architecture of MuvB assembly and the crystal structure of a subcomplex critical for MuvB function in gene repression. We find that the MuvB subunits LIN9 and LIN37 function as scaffolding proteins that arrange the other subunits LIN52, LIN54 and RBAP48 for TF, DNA, and histone binding, respectively. Biochemical and structural data demonstrate that MuvB binds nucleosomes through an interface that is distinct from LIN54-DNA consensus site recognition and that MuvB increases nucleosome occupancy in a reconstituted promoter. We find in arrested cells that MuvB primarily associates with a tightly positioned +1 nucleosome near the transcription start site (TSS) of MuvB-regulated genes. These results support a model that MuvB binds and stabilizes nucleosomes just downstream of the TSS on its target promoters to repress gene expression.

The chromatin architecture in promoters is thought to regulate gene expression, but it remains uncertain how most transcription factors (TFs) impact nucleosome position. The MuvB TF complex regulates cell-cycle dependent gene-expression and is critical for differentiation and proliferation during development and cancer. MuvB can both positively and negatively regulate expression, but the structure of MuvB and its biochemical function are poorly understood. Here we determine the overall architecture of MuvB assembly and the crystal structure of a subcomplex critical for MuvB function in gene repression. We find that the MuvB subunits LIN9 and LIN37 function as scaffolding proteins that arrange the other subunits LIN52, LIN54 and RBAP48 for TF, DNA, and histone binding, respectively. Biochemical and structural data demonstrate that MuvB binds nucleosomes through an interface that is distinct from LIN54-DNA consensus site recognition and that MuvB increases nucleosome occupancy in a reconstituted promoter. We find in arrested cells that MuvB primarily associates with a tightly positioned +1 nucleosome near the transcription start site (TSS) of MuvB-regulated genes. These results support a model that MuvB binds and stabilizes nucleosomes just downstream of the TSS on its target promoters to repress gene-expression.

Transcription occurs in stochastic bursts, i.e., transcription events are temporally clustered. The clustering does not ensue from environmental fluctuations but springs from the intrinsically stochastic behavior of the regulatory process that controls transcription. Based on microscopic observations of transcription at a single gene copy of yeast, we show that the regulatory process is cyclic and irreversible, i.e., the process violates the detailed balance conditions for thermodynamic equilibrium. The theoretical significance of this finding is discussed.