Oligomerization and binding of the Dnmt3a DNA methyltransferase to parallel DNA molecules: heterochromatic localization and role of Dnmt3L.

Structural studies showed that Dnmt3a has two interfaces for protein-protein interaction in the heterotetrameric Dnmt3a/3L C-terminal domain complex: the RD interface (mediating the Dnmt3a-3a contact) and the FF interface (mediating the Dnmt3a-3L contact). Here, we demonstrate that Dnmt3a-C forms dimers via the FF interface as well, which further oligomerize via their RD interfaces. Each RD interface of the Dnmt3a-C oligomer creates an independent DNA binding site, which allows for binding of separate DNA molecules oriented in parallel.

Photocaged variants of the MunI and PvuII restriction enzymes.

Regulation of proteins by light is a new and promising strategy for the external control of biological processes. In this study, we demonstrate the ability to regulate the catalytic activity of the MunI and PvuII restriction endonucleases with light. We used two different approaches to attach a photoremovable caging compound, 2-nitrobenzyl bromide (NBB), to functionally important regions of the two enzymes.

A flexible bipartite coiled coil structure is required for the interaction of Hexim1 with the P-TEFB subunit cyclin T1.

Transcription elongation is regulated by the cellular protein Hexim1, which inhibits phosphorylation of RNA polymerase II by interacting with the positive transcription elongation factor P-TEFb. Hexim1 binds directly to Cyclin T1 of P-TEFb with its coiled coil domain that is subdivided into a highly polar N-terminal segment containing nonconservative residues in the dimer interface and a C-terminal segment with an evolutionarily conserved sequence composition. Here we show that the noncanonical sequence composition of the first coiled coil segment is required for the interaction with Cyclin T1 while the second segment keeps the Cyclin T-binding domain dimeric upon binding.

The C-terminal domain is sufficient for endonuclease activity of Neisseria gonorrhoeae MutL.

The mutL gene of Neisseria gonorrhoeae has been cloned and the gene product purified. We have found that the homodimeric N. gonorrhoeae MutL (NgoL) protein displays an endonuclease activity that incises covalently closed circular DNA in the presence of Mn(2+), Mg(2+) or Ca(2+) ions, unlike human MutLalpha which shows endonuclease activity only in the presence of Mn(2+).

Formation of nucleoprotein filaments by mammalian DNA methyltransferase Dnmt3a in complex with regulator Dnmt3L.

The C-terminal domains of Dnmt3a and Dnmt3L form elongated heterotetramers (3L-3a-3a-3L). Analytical ultracentrifugation confirmed the Dnmt3a-C/3L-C complex exists as a 2:2 heterotetramer in solution. The 3a-3a interface is the DNA-binding site, while both interfaces are essential for AdoMet binding and catalytic activity.

Clustering of VASP actively drives processive, WH2 domain-mediated actin filament elongation.

Vasodilator-stimulated phosphoprotein (VASP) is a key regulator of dynamic actin structures like filopodia and lamellipodia, but its precise function in their formation is controversial. Using in vitro TIRF microscopy, we show for the first time that both human and Dictyostelium VASP are directly involved in accelerating filament elongation by delivering monomeric actin to the growing barbed end. In solution, DdVASP markedly accelerated actin filament elongation in a concentration-dependent manner but was inhibited by low concentrations of capping protein (CP).

Single-stranded DNA-binding protein recruits DNA polymerase V to primer termini on RecA-coated DNA.

Translesion DNA synthesis (TLS) by DNA polymerase V (polV) in Escherichia coli involves accessory proteins, including RecA and single-stranded DNA-binding protein (SSB). To elucidate the role of SSB in TLS we used an in vitro exonuclease protection assay and found that SSB increases the accessibility of 3' primer termini located at abasic sites in RecA-coated gapped DNA. The mutant SSB-113 protein, which is defective in protein-protein interactions, but not in DNA binding, was as effective as wild-type SSB in increasing primer termini accessibility, but deficient in supporting polV-catalyzed TLS.

Tetrameric restriction enzymes: expansion to the GIY-YIG nuclease family.

The GIY-YIG nuclease domain was originally identified in homing endonucleases and enzymes involved in DNA repair and recombination. Many of the GIY-YIG family enzymes are functional as monomers. We show here that the Cfr42I restriction endonuclease which belongs to the GIY-YIG family and recognizes the symmetric sequence 5'-CCGC/GG-3' ('/' indicates the cleavage site) is a tetramer in solution.

Monomeric restriction endonuclease BcnI in the apo form and in an asymmetric complex with target DNA.

Restriction endonuclease BcnI cleaves duplex DNA containing the sequence CC/SGG (S stands for C or G, / designates a cleavage position) to generate staggered products with single nucleotide 5'-overhangs. Here, we show that BcnI functions as a monomer that interacts with its target DNA in 1:1 molar ratio and report crystal structures of BcnI in the absence and in the presence of DNA. In the complex with DNA, BcnI makes specific contacts with all five bases of the target sequence and not just with a half-site, as the protomer of a typical dimeric restriction endonuclease.

Sulfate acts as phosphate analog on the monomeric catalytic fragment of the CPx-ATPase CopB from Sulfolobus solfataricus.

The crystal structure of the catalytic fragment of a Sulfolobus solfataricus P-type ATPase, CopB-B, was determined with a 2.6 A resolution. CopB-B is the major soluble fragment of the archaeal CPx-ATPase CopB and is comprized of a nucleotide and a phosphorylation domain.

Restriction endonuclease BpuJI specific for the 5'-CCCGT sequence is related to the archaeal Holliday junction resolvase family.

Type IIS restriction endonucleases (REases) recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. REase BpuJI recognizes the asymmetric sequence 5'-CCCGT, however it cuts at multiple sites in the vicinity of the target sequence. We show that BpuJI is a dimer, which has two DNA binding surfaces and displays optimal catalytic activity when bound to two recognition sites.

Restriction endonuclease MvaI is a monomer that recognizes its target sequence asymmetrically.

Restriction endonuclease MvaI recognizes the sequence CC/WGG (W stands for A or T, '/' designates the cleavage site) and generates products with single nucleotide 5'-overhangs. The enzyme has been noted for its tolerance towards DNA modifications. Here, we report a biochemical characterization and crystal structures of MvaI in an apo-form and in a complex with target DNA at 1.

3D structure of Thermus aquaticus single-stranded DNA-binding protein gives insight into the functioning of SSB proteins.

In contrast to the majority of tetrameric SSB proteins, the recently discovered SSB proteins from the Thermus/Deinoccus group form dimers. We solved the crystal structures of the SSB protein from Thermus aquaticus (TaqSSB) and a deletion mutant of the protein and show the structure of their ssDNA binding domains to be similar to the structure of tetrameric SSBs. Two conformations accompanied by proline cis-trans isomerization are observed in the flexible C-terminal region.

Structural and functional analysis of the MutS C-terminal tetramerization domain.

The Escherichia coli DNA mismatch repair (MMR) protein MutS is essential for the correction of DNA replication errors. In vitro, MutS exists in a dimer/tetramer equilibrium that is converted into a monomer/dimer equilibrium upon deletion of the C-terminal 53 amino acids. In vivo and in vitro data have shown that this C-terminal domain (CTD, residues 801-853) is critical for tetramerization and the function of MutS in MMR and anti-recombination.

Allosteric communication network in the tetrameric restriction endonuclease Bse634I.

Restriction endonuclease Bse634I is a homotetramer arranged as a dimer of two primary dimers. Bse634I displays its maximum catalytic efficiency upon binding of two copies of cognate DNA, one per each primary dimer. The catalytic activity of Bse634I on a single DNA copy is down-regulated due to the cross-talking interactions between the primary dimers.

The crystal structure of the rare-cutting restriction enzyme SdaI reveals unexpected domain architecture.

Rare-cutting restriction enzymes are important tools in genome analysis. We report here the crystal structure of SdaI restriction endonuclease, which is specific for the 8 bp sequence CCTGCA/GG ("/" designates the cleavage site). Unlike orthodox Type IIP enzymes, which are single domain proteins, the SdaI monomer is composed of two structural domains.

Biochemical indication for myristoylation-dependent conformational changes in HIV-1 Nef.

The accessory HIV-1 Nef protein is essential for viral replication, high virus load, and progression to AIDS. These functions are mediated by the alteration of signaling and trafficking pathways and require the membrane association of Nef by its N-terminal myristoylation. However, a large portion of Nef is also found in the cytosol, in line with the observation that myristoylation is only a weak lipidation anchor for membrane attachment.

Sedimentation velocity method in the analytical ultracentrifuge for the study of protein-protein interactions.

Sedimentation analysis in the analytical ultracentrifuge can be employed to detect macromolecular interactions. Whenever two molecules interact the mass of the resulting complex is increased and this is reflected in the sedimentation behavior. In this chapter we discuss how this phenomenon can be utilized to determine quantitative parameters of an interaction.

Sedimentation velocity analysis of heterogeneous protein-protein interactions: Lamm equation modeling and sedimentation coefficient distributions c(s).

We describe algorithms for solving the Lamm equations for the reaction-diffusion-sedimentation process in analytical ultracentrifugation, and examine the potential and limitations for fitting experimental data. The theoretical limiting case of a small, uniformly distributed ligand rapidly reacting with a larger protein in a "constant bath" of the ligand is recapitulated, which predicts the reaction boundary to sediment with a single sedimentation and diffusion coefficient. As a consequence, it is possible to express the sedimentation profiles of reacting systems as c(s) distribution of noninteracting Lamm equation solutions, deconvoluting the effects of diffusion.

Single-stranded DNA-binding protein of Deinococcus radiodurans: a biophysical characterization.

The highly conserved bacterial single-stranded DNA-binding (SSB) proteins play an important role in DNA replication, repair and recombination and are essential for the survival of the cell. They are functional as tetramers, in which four OB(oligonucleotide/oligosaccharide binding)-folds act as DNA-binding domains. The protomer of the SSB protein from the extremely radiation-resistant organism Deinococcus radiodurans (DraSSB) has twice the size of the other bacterial SSB proteins and contains two OB-folds.

A novel octameric AMP-forming acetyl-CoA synthetase from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum.

AMP-forming acetyl-CoA synthetases (ACSs) are ubiquitous in all three domains of life. Here, we report the first characterization of an ACS from a hyperthermophilic organism, from the archaeon Pyrobaculum aerophilum. The recombinant ACS, the gene product of ORF PAE2867, showed extremely high thermostability and thermoactivity at temperatures around 100 degrees C.

Crystal structure and interactions of the PAS repeat region of the Drosophila clock protein PERIOD.

PERIOD proteins are central components of the Drosophila and mammalian circadian clock. Their function is controlled by daily changes in synthesis, cellular localization, phosphorylation, degradation, as well as specific interactions with other clock components. Here we present the crystal structure of a Drosophila PERIOD (dPER) fragment comprising two tandemly organized PAS (PER-ARNT-SIM) domains (PAS-A and PAS-B) and two additional C-terminal alpha helices (alphaE and alphaF).

Complex formation and catalytic activation by the PII signaling protein of N-acetyl-L-glutamate kinase from Synechococcus elongatus strain PCC 7942.

The signal transduction protein P(II) from the cyanobacterium Synechococcus elongatus strain PCC 7942 forms a complex with the key enzyme of arginine biosynthesis, N-acetyl-l-glutamate kinase (NAGK). Here we report the effect of complex formation on the catalytic properties of NAGK. Although pH and ion dependence are not affected, the catalytic efficiency of NAGK is strongly enhanced by binding of P(II), with K(m) decreasing by a factor of 10 and V(max) increasing 4-fold.

Bifunctional phosphoglucose/phosphomannose isomerase from the hyperthermophilic archaeon Pyrobaculum aerophilum.

ORF PAE1610 from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum was first annotated as the conjectural pgi gene coding for hypothetical phosphoglucose isomerase (PGI). However, we have recently identified this ORF as the putative pgi/pmi gene coding for hypothetical bifunctional phosphoglucose/phosphomannose isomerase (PGI/PMI). To prove its coding function, ORF PAE1610 was overexpressed in Escherichia coli, and the recombinant enzyme was characterized.