Physical association and coordinate function of the H3 K4 methyltransferase MLL1 and the H4 K16 acetyltransferase MOF.

A stable complex containing MLL1 and MOF has been immunoaffinity purified from a human cell line that stably expresses an epitope-tagged WDR5 subunit. Stable interactions between MLL1 and MOF were confirmed by reciprocal immunoprecipitation, cosedimentation, and cotransfection analyses, and interaction sites were mapped to MLL1 C-terminal and MOF zinc finger domains. The purified complex has a robust MLL1-mediated histone methyltransferase activity that can effect mono-, di-, and trimethylation of H3 K4 and a MOF-mediated histone acetyltransferase activity that is specific for H4 K16.

Proteomic and genomic characterization of chromatin complexes at a boundary.

We have dissected specialized assemblies on the Saccharomyces cerevisiae genome that help define and preserve the boundaries that separate silent and active chromatin. These assemblies contain characteristic stretches of DNA that flank particular regions of silent chromatin, as well as five distinctively modified histones and a set of protein complexes. The complexes consist of at least 15 chromatin-associated proteins, including DNA pol epsilon, the Isw2-Itc1 and Top2 chromatin remodeling proteins, the Sas3-Spt16 chromatin modifying complex, and Yta7, a bromodomain-containing AAA ATPase.

Multiple full-length NS3 molecules are required for optimal unwinding of oligonucleotide DNA in vitro.

NS3 (nonstructural protein 3) from the hepatitis C virus is a 3' --> 5' helicase classified in helicase superfamily 2. The optimally active form of this helicase remains uncertain. We have used unwinding assays in the presence of a protein trap to investigate the first cycle of unwinding by full-length NS3.

Genetic and biochemical evaluation of the importance of Cdc6 in regulating mitotic exit.

We evaluated the hypothesis that the N-terminal region of the replication control protein Cdc6 acts as an inhibitor of cyclin-dependent kinase (Cdk) activity, promoting mitotic exit. Cdc6 accumulation is restricted to the period from mid-cell cycle until the succeeding G1, due to proteolytic control that requires the Cdc6 N-terminal region. During late mitosis, Cdc6 is present at levels comparable with Sic1 and binds specifically to the mitotic cyclin Clb2.

Pre-steady-state DNA unwinding by bacteriophage T4 Dda helicase reveals a monomeric molecular motor.

Helicases are molecular motor enzymes that unwind and translocate nucleic acids. One of the central questions regarding helicase activity is whether the process of coupling ATP hydrolysis to DNA unwinding requires an oligomeric form of the enzyme. We have applied a pre-steady-state kinetics approach to address this question with the bacteriophage T4 Dda helicase.

Non-Watson-Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase.

Peptide nucleic acid (PNA) is a DNA mimic in which the nucleobases are linked by an N-(2-aminoethyl) glycine backbone. Here we report that PNA can interact with single-stranded DNA (ssDNA) in a non-sequence-specific fashion. We observed that a 15mer PNA inhibited the ssDNA-stimulated ATPase activity of a bacteriophage T4 helicase, Dda.

Hepatitis C virus NS3 and simian virus 40 T antigen helicases displace streptavidin from 5'-biotinylated oligonucleotides but not from 3'-biotinylated oligonucleotides: evidence for directional bias in translocation on single-stranded DNA.

Helicases are enzymes that use energy from nucleoside triphosphate hydrolysis to unwind double-stranded (ds) DNA, a process vital to virtually every phase of DNA metabolism. Helicases have been classified as either 5'-to-3' or 3'-to-5' on the basis of their ability to unwind duplex DNA adjacent to either a 5' or 3' single-stranded (ss) DNA overhang. However, there has been debate as to whether this substrate preference is indicative of unidirectional translocation on ssDNA.

Measurement of steady-state kinetic parameters for DNA unwinding by the bacteriophage T4 Dda helicase: use of peptide nucleic acids to trap single-stranded DNA products of helicase reactions.

Measurement of steady-state rates of unwinding of double-stranded oligonucleotides by helicases is hampered due to rapid reannealing of the single-stranded DNA products. Including an oligonucleotide in the reaction mixture which can hybridize with one of the single strands can prevent reannealing. However, helicases bind to single-stranded DNA, therefore the additional oligonucleotide can sequester the enzyme, leading to slower observed rates for unwinding.

Evidence for a functional monomeric form of the bacteriophage T4 DdA helicase. Dda does not form stable oligomeric structures.

The active form of many helicases is oligomeric, possibly because oligomerization provides multiple DNA binding sites needed for unwinding of DNA. In order to understand the mechanism of the bacteriophage T4 Dda helicase, the potential requirement for oligomerization was investigated. Chemical cross-linking and high pressure gel filtration chromatography provided little evidence for the formation of an oligomeric species.

Biotin-streptavidin-labeled oligonucleotides as probes of helicase mechanisms.

Helicases use the energy from ATP hydrolysis to catalyze formation of single-stranded nucleic acids by unwinding double-stranded nucleic acids. The ATP-dependent reaction can be broken down into at least two steps: melting of the duplex and translocation of the enzyme along the nucleic acid lattice. Each step presents difficulties for study because clear end points for the reactions are not always available.

Unwinding of unnatural substrates by a DNA helicase.

Helicases separate double-stranded DNA into single-stranded DNA intermediates that are required during replication and recombination. These enzymes are believed to transduce free energy available from ATPase activity to unwind the duplex and translocate along the nucleic acid lattice. The nature of enzyme-substrate interactions between helicases and duplex DNA substrates has not been well-defined.

Unwinding of nucleic acids by HCV NS3 helicase is sensitive to the structure of the duplex.

Hepatitis C virus (HCV) helicase, non-structural protein 3 (NS3), is proposed to aid in HCV genome replication and is considered a target for inhibition of HCV. In order to investigate the substrate requirements for nucleic acid unwinding by NS3, substrates were prepared by annealing a 30mer oligonucleotide to a 15mer. The resulting 15 bp duplex contained a single-stranded DNA overhang of 15 nt referred to as the bound strand.

Results of staged bilateral carotid endarterectomy.

To determine differences in outcome between unilateral and staged bilateral carotid endarterectomies, we reviewed 850 carotid endarterectomies done by 14 surgeons in a community hospital. Results of 528 unilateral procedures were compared with those of 161 bilateral procedures. Data were abstracted from records for an 11-year period.

Abdominal aortic aneurysm infected with Campylobacter fetus subspecies fetus.

We report a survivor of Campylobacter fetus septicemia from an infected abdominal aortic aneurysm who was successfully treated with an anatomic graft reconstruction and antibiotics. According to a survey of the English-language medical literature this was the fourth such patient successfully treated. C.

Infrequency of blacks among patients having carotid endarterectomy.

We reviewed demographic data on patients having 2,256 carotid endarterectomies in eight large hospitals in North Carolina to determine the frequency of blacks among these patients. Blacks comprised only 4.6% of the patients having carotid endarterectomy even though they comprised 26% of all patients discharged and 22% of the general population of the state.

Vascular injuries to the extremities.

The treatment of vascular trauma is a challenging and increasingly successful endeavor. Improvement in patency rates and limb salvage have occurred through advances in diagnostic methods, materials for vessel replacement and techniques of repair. Several principals have evolved.