Biomedical subjects

T J Fu

Publications and source records attributed to T J Fu.

6 recordsLinked to original sources

PubMed1996

A direct interaction between a DNA-tracking protein and a promoter recognition protein: implications for searching DNA sequence.

Bacteriophage T4 gene 45 protein, gp45, serves as the sliding clamp of viral DNA replication and as the activator of T4 late gene transcription. In the latter context, DNA tracking is an essential feature of the unique mechanism of action. T4 late promoters, which consist of a simple TATA box, TATAAATA, are recognized by the small sigma-family gene 55 protein, gp55, which binds to Escherichia coli RNA polymerase core. A direct and RNA polymerase-independent interaction of gp45 with gp55 has been demonstrated in two ways. (i) gp45 tracks along DNA; co-tracking of gp55 requires the previously documented DNA-loading process of gp45, and can be detected by photochemical crosslinking. (ii) The dynamics of DNA tracking by gp45 can be followed by footprinting; the catenated DNA-tracking state of gp45 is short-lived, but is stabilized by gp55. The ability of this topologically linked DNA-tracking transcriptional activator to interact directly with a promoter recognition protein suggests the existence of multiple pathways of promoter location, which are discussed.

Bacterial Proteins↗

PubMed1996

Dynamics of DNA-tracking by two sliding-clamp proteins.

Bacteriophage T4 gene 45 protein (gp45) and Escherichia coli beta are DNA-tracking sliding-clamp proteins that increase processivity by tethering their conjugate DNA polymerases to DNA. gp45 also activates T4 late transcription. DNA loading of gp45 and beta requires ATP or dATP hydrolysis; efficient loading at primer-template junctions is assisted by single-stranded DNA-binding proteins. The kinetics of gp45 loading and tracking have been examined by DNase I footprinting of linear DNA with one blunt end, one primer-template junction, and binding sites for proteins that block gp45 tracking. DNA loading of gp45 can also be interrupted by adding the non-hydrolyzable ATP analog ATP-gamma-S. At saturation, DNA is very closely packed with gp45 or beta. When gp45 loading is interrupted, or when a segment of the track is blocked off, the gp45 footprint dissipates within seconds, but the DNA-tracking state of beta is much more stable. The stability of the tracking state of gp45 is, however, increased by the macromolecular crowding agent polyethylene glycol. We suggest that labile gp45 catenation directly generates the coupling of late transcription to DNA replication during bacteriophage T4 multiplication.

Adenosine Triphosphate↗

PubMed1994

Cleavage of double-crossover molecules by T4 endonuclease VII.

DNA double-crossover molecules containing two Holliday junctions have been prepared and treated with endonuclease VII, the resolvase from bacteriophage T4. One molecule contains antiparallel double-helical domains, and the other molecule contains parallel domains. The parallel double-crossover model system has been made tractable by closing the free ends of the molecule, to convert it to a catenane. The products resulting from the two substrates differ substantially. The molecule containing antiparallel helical domains is cleaved three nucleotides 3' to the crossover points, in a fashion similar to single Holliday junction analogs. The molecule containing parallel helical domains is cleaved, but the major points of scission are five nucleotides 5' to a branch point on the crossover strands and six nucleotides 3' to the same branch point on the non-crossover strands. The major sites of scission reflect features of molecular symmetry in each case, suggesting that the resolvase recognizes structural features. The cleavage results suggest that the antiparallel structure is the natural substrate, if the Holliday junction is unconstrained within the cell. It is straightforward to reconcile antiparallel Holliday junctions with the conventional parallel paradigm of recombination. Nevertheless, the cleavage of the parallel molecule shows that a parallel substrate could also be cleaved symmetrically by endonuclease VII (but with different products) if the molecule were constrained to assume that conformation within the cell.

Base Sequence↗

PubMed1994

Holliday junction crossover topology.

The Holliday junction is a key intermediate in genetic recombination. It consists of four strands of DNA that associate to form four double-helical arms. Studies over the past decade with asymmetric analogs of Holliday junctions have shown that the four arms stack to generate two stacking domains. This arrangement of arms results in two strands with a roughly helical structure, and two that contain a crossover structure connecting the domains. Both parallel and antiparallel orientations of the helical strands are possible, although the antiparallel orientation is favored. In principle, there are two possible isomers of the Holliday junction, depending on which pairs of strands contain the crossover structure; isomerization between these two structures is key to many molecular models of recombination. Isomerization of parallel domain structures entails large end-to-end helical rotations if braiding of the crossover strands is to be avoided. We have examined the ability of the crossover strands to braid. This has been done by employing a double crossover molecule, whose termini are all hairpin loops. Such a molecule is a catenane of two single strands of DNA, whose linking number is a function of the sign of the node at the crossover. We have prepared linking standards by means of topological protection techniques, and have compared them to the catenane formed by the double crossover molecule. We find no evidence for braiding of the strands. Furthermore, no braided structures can be detected when the double crossover molecule is treated with Escherichia coli DNA topoisomerase I in the presence of varying amounts of Mg2+ cations.

Base Sequence↗

PubMed1993

Symmetric immobile DNA branched junctions.

Branch migration is an isomerization of Holliday recombination intermediates that arises from their homologous (2-fold) sequence symmetry. This isomerization relocates the branch point in an apparently random fashion and thereby complicates the study of the physical and structural properties of these structures. For the past decade, these properties have been studied in low-symmetry immobile junctions, whose sequence asymmetry eliminates branch migration. The asymmetric findings of many of these studies suggest the need for a system combining both immobility and symmetry. Double-crossover DNA molecules have been used to create molecules with both these properties. Immobility is achieved by flanking one crossover with a symmetric junction and the other crossover with an asymmetric junction. Close torsional coupling between the two junctions renders the symmetric junction immobile. These molecules will enable the characterization of thermodynamic, structural, dynamic, liganding, and substrate properties of symmetric branched DNA molecules in a sequence-specific fashion.

Base Sequence↗

PubMed1993

DNA double-crossover molecules.

DNA molecules containing two crossover sites between helical domains have been suggested as intermediates in recombination processes involving double-strand breaks. We have modeled these double-crossover structures in an oligonucleotide system. Whereas the relative orientations of the helical domains must be specified in designing these molecules, there are two broad classes of the molecules, the parallel, DP, and antiparallel, DA, molecules. The distance between crossover points must be specified as multiples of half-turns, in order to avoid torsional stress in this system; hence, there are two further subdivisions, those double-crossover molecules separated by odd, O, and even, E, numbers of half-turns. In addition, the parallel molecules with odd numbers of half-turns between crossovers must be divided into those with an excess major or wide-groove separation, W, or those with an excess minor- or narrow-groove separation, N. We have constructed models of all five of these classes, DAE, DAO, DPE, DPOW, and DPON. DPE molecules containing 1 and 2 helical turns between crossovers have been constructed; the DAE molecule contains 1 turn between crossovers, and the DAO, DPOW, and DPON molecules contain 1.5 helical turns between crossovers. None of the parallel molecules is well-behaved; the molecules either dissociate or form multimers when visualized on native polyacrylamide gels. In contrast, antiparallel molecules form single bands when assayed in this fashion. Hydroxyl radical autofootprinting analysis of these molecules reveals protection at expected sites of crossover and of occlusion, suggesting that all the complexes contain linear helix axes that are roughly coplanar between crossovers. However, the DPOW molecule and the DPE molecule with 2 turns between crossovers show decreased protection in the portion between crossovers, suggesting that their helices may bow in response to charge repulsion. We conclude that the helices between parallel double crossovers must be shielded from each other or distorted from linearity if they are to participate in recombination. We have analyzed the possibilities of branch migration and crossover isomerization in double-crossover molecules. Parallel molecules need no sequence symmetry beyond homology to branch migrate, but the sequence symmetry requirements for antiparallel molecules restrict migration to directly repetitive segments that iterate the sequence between crossovers. Crossover isomerization appears to be a very complex process in parallel double-crossover molecules, suggesting that it may be catalyzed by topoisomerases if it occurs within the cell.

Base Sequence↗