A new approach to ultrasensitive detection of DNA hybridization based on nanoparticle-amplified surface plasmon resonance (SPR) is described. Use of the Au nanoparticle tags leads to a greater than 10-fold increase in angle shift, corresponding to a more than 1000-fold improvement in sensitivity for the target oligonucleotide as compared to the unamplified binding event. This enhanced shift in SPR reflectivity is a combined result of greatly increased surface mass, high dielectric constant of Au particles, and electromagnetic coupling between Au nanoparticles and the Au film. DNA melting and digestion experiments further supported the feasibility of this approach in DNA hybridization studies. The extremely large angle shifts observed in particle-amplified SPR make it possible to conduct SPR imaging experiments on DNA arrays. In the present work, macroscopic 4 × 4 arrays were employed, and a ∼10 pM limit of quantitation was achieved for 24-mer oligonucleotides (surface density ≤8 × 108 molecules/cm2). Even without further optimization, the sensitivity of this technique begins to approach that of traditional fluorescence-based methods for DNA hybridization. These results illustrate the potential of particle-amplified SPR for array-based DNA analysis and ultrasensitive detection of oligonucleotides.

The elaborate process of genomic replication requires a large collection of proteins properly assembled at a DNA replication fork. Several decades of research on the bacterium Escherichia coli and its bacteriophages T4 and T7 have defined the roles of many proteins central to DNA replication. These three different prokaryotic replication systems use the same fundamental components for synthesis at a moving DNA replication fork even though the number and nature of some individual proteins are different and many lack extensive sequence homology. The components of the replication complex can be grouped into functional categories as follows: DNA polymerase, helix destabilizing protein, polymerase accessory factors, and primosome (DNA helicase and DNA primase activities). The replication of DNA derives from a multistep enzymatic pathway that features the assembly of accessory factors and polymerases into a functional holoenzyme; the separation of the double-stranded template DNA by helicase activity and its coupling to the primase synthesis of RNA primers to initiate Okazaki fragment synthesis; and the continuous and discontinuous synthesis of the leading and lagging daughter strands by the polymerases. This review summarizes and compares and contrasts for these three systems the types, timing, and mechanism of reactions and of protein-protein interactions required to initiate, control, and coordinate the synthesis of the leading and lagging strands at a DNA replication fork and comments on their generality.

The DNA replication complex of bacteriophage T4 has been assembled as a single unit on a minicircle substrate with a replication fork that permits an independent measurement of the amount of DNA synthesis on both the leading and lagging strands. The assembled replisome consists of the T4 polymerase [gene product 43 (gp43)], clamp protein (gp45), clamp loader (gp44͞62), helicase (gp41), helicase accessory factor (gp59), primase (gp61), and singlestranded DNA binding protein (gp32). We demonstrate that on the minicircle the synthesis of the leading and lagging strands are coordinated and that the C-terminal domain of the gp32 protein regulates this coordination. We show that the reconstituted replisome encompasses two coupled holoenzyme complexes and present evidence that this coupling might include a gp43 homodimer interaction. Bacteriophage T4 is one of the more elementary replication systems using eight proteins in the formation and propagation of a replication fork. Central to the replication process, the T4 DNA polymerase [gene product 43 (gp43)] catalyzes nucleotide incorporation in a 5Ј to 3Ј direction whereas its 3Ј to 5Ј exonuclease activity serves to maintain fidelity during replication (1). Alone, the T4 polymerase will incorporate nucleotides in a distributive fashion, and, to avert quick dissociation from the DNA, interactions between the gp43 polymerase and accessory factors are essential (2, 3). These accessory factors include the gp45 protein (sliding clamp), which possesses a ring-shaped structure with an internal diameter large enough to encircle DNA, and the 44͞62 protein complex, which loads the sliding clamp onto DNA (4, 5). In addition, a DNA helicase (gene product 41) is required for the ATP-or GTP-dependent unwinding of the duplex DNA to be replicated at the front of the replication fork (6). The T4 single-stranded DNA binding protein gp32 is needed to prevent the reannealing of duplex DNA at the replication fork (7). The helicase has its own accessory factor, the gene product 59 protein, which is required to load the gp41 helicase onto single-stranded DNA coated with the gp32 single-stranded DNA binding protein (8). Finally, associated with the helicase is the DNA primase (gp61), which provides the pentaribonucleotide primers needed for the initiation of laggingstrand DNA synthesis (9).The dynamics of holoenzyme (gp43, gp45, and gp44͞62) assembly have been addressed both kinetically and structurally (10). The holoenzyme assembly process proceeds in a ordered fashion in which the gp45 sliding clamp is first loaded onto the DNA by the 44͞62 protein complex in an ATP-dependent process followed by the rapid association of the polymerase (gp43), with the 45 clamp mediated through the carboxyl terminus of the polymerase (11, 12). Once formed, the holoenzyme is stable with a dissociation rate constant (k off ) of 0.01 sec Ϫ1. The same basic replication strategy is shared by all DNA polymerases characterized to date in which DNA synthesis occurs by the successive addition of nucleotides to the 3Ј ...

Homologous strand exchange is a central step in general genetic recombination. A multiprotein complex composed of five purified bacteriophage T4 proteins (the products of the uvsX, uvsY, 32, 41, and 59 genes) that mediates strand exchange under physiologically relevant conditions has been reconstituted. One of these proteins, the product of the uvsY gene, is required for homologous pairing but strongly inhibits branch migration catalyzed by UvsX protein, the phage RecA analog. Branch migration is completely dependent on the gene 41 protein, a DNA helicase that also functions in phage replication. The helicase is delivered to the strand exchange complex by the gene 59 accessory protein in a strand-specific fashion through direct interactions between the gene 59 and gene 32 proteins. These data suggest that strand transferases such as UvsX protein are essential for homologous pairing in vivo, but that a DNA helicase drives polar branch migration.

Recent methods to create large libraries of proteins have greatly advanced the discovery of proteins with novel functions. However, one limitation in the discovery of new biocatalysts is the screening or selection methods employed to find enzymes from these libraries. We have developed a potentially general method termed QUEST (QUerying for EnzymeS using the Three-hybrid system), which allows the construction of an easily screened or selected phenotype for, in theory, any type of enzymatic reaction. The method couples the in vivo concentration of an enzyme's substrate to changes in the transcriptional level of a reporter operon. Using the arabinose operon activator AraC, we constructed a system capable of detecting the fungal enzyme scytalone dehydratase (SD) in bacteria, and demonstrated its sensitivity and usefulness in library screening.