Single-stranded DNA Binding Protein Gp32 Research Articles

Isothermal Amplification of Long, Discrete DNA Fragments Facilitated by Single-Stranded Binding Protein

Isothermal amplification methods for detection of DNA and RNA targets have expanded significantly in recent years, promising a new wave of simple and rapid molecular diagnostics. Current isothermal methods result in the generation of short fragments (<150 base pairs) or highly branched long DNA products. Here we report the amplification of discrete target fragments of several kilobases at 37 °C from both double- and single-stranded circular template DNA using specific primer pairs. In contrast to existing methods, this amplification requires only the single-stranded DNA-binding protein gp32 from bacteriophage T4 and a strand-displacing DNA polymerase. In addition to the discrete amplicon products, this method also produces higher molecular weight products consisting of multiple repeated copies of the amplicon and template DNA. We demonstrate that two features of gp32 enable this amplification: a facilitation of primer strand invasion into double-stranded DNA, and a suppression of non-homologous primer annealing and nonspecific amplification. The ability presented here to produce long, discrete DNA products in an isothermal reaction extends the scope of isothermal amplification to enable more useful applications of these promising methods.

Interaction of T4 UvsW Helicase and Single-Stranded DNA Binding Protein gp32 through Its Carboxy-Terminal Acidic Tail

Bacteriophage T4 UvsW helicase contains both unwinding and annealing activities and displays some functional similarities to bacterial RecG and RecQ helicases. UvsW is involved in several DNA repair pathways, playing important roles in recombination-dependent DNA repair and the reorganization of stalled replication forks. The T4 single-stranded DNA (ssDNA) binding protein gp32 is a central player in nearly all DNA replication and repair processes and is thought to facilitate their coordination by recruiting and regulating the various proteins involved. Here, we show that the activities of the UvsW protein are modulated by gp32. UvsW-catalyzed unwinding of recombination intermediates such as D-loops and static X-DNA (Holliday junction mimic) to ssDNA products is enhanced by the gp32 protein. The enhancement requires the presence of the protein interaction domain of gp32 (the acidic carboxy-terminus), suggesting that a specific interaction between UvsW and gp32 is required. In the absence of this interaction, the ssDNA annealing and ATP-dependent translocation activities of UvsW are severely inhibited when gp32 coats the ssDNA lattice. However, when UvsW and gp32 do interact, UvsW is able to efficiently displace the gp32 protein from the ssDNA. This ability of UvsW to remove gp32 from ssDNA may explain its ability to enhance the strand invasion activity of the T4 recombinase (UvsX) and suggests a possible new role for UvsW in gp32-mediated DNA transactions.

A Novel Mechanism of Virus–Virus Interactions: Bacteriophage P2 Tin Protein Inhibits Phage T4 DNA Synthesis by Poisoning the T4 Single-Stranded DNA Binding Protein, gp32

P2 prophages have been known to inhibit DNA replication and growth of T-even phages. We show here that this inhibition is due to poisoning of the T-even single-stranded DNA binding protein gp32 by the product of the nonessential P2tingene. Synthesis of Tin protein from a gene cloned in a multicopy plasmid is necessary and sufficient to completely preventde novoDNA replication and growth of wild-type T2 or T4 phage. We isolated more than 20 independent mutants that render T-even phages resistant to poisoning by the P2 Tin protein. In all of these mutants, which we callasp,Asp codon 163 of gene32is changed to a Gly or Asn codon. The mutant alleles are recessive; i.e., when wild-type andaspmutants coinfect the same host cells, most DNA replication is poisoned by P2 Tin protein. To explain our results, we propose that the P2 Tin protein interacts with T-even gp32 at position 163 and distorts the helical filament of gene32protein on single-stranded DNA. Thereby Tin protein inhibits either assembly or function, or both, of the T4 replisome. The inhibition of late gene expression by P2 Tin protein may be an indirect consequence of inhibition of DNA replication.

Complex between single-stranded DNA and gene 5 protein of bacteriophage M13 studied with linear dichroism and ultraviolet absorption

We have studied complexes between the gene 5 protein (gp5) of bacteriophage M13 and various polynucleotides, including single-stranded DNA, using ultraviolet absorption and linear dichroism. Upon complex formation the absorption spectra of both the protein and the polynucleotides change. The protein absorption changes indicate that for at least two of the five tyrosine residues per protein monomer the environment becomes less polar upon binding to the polynucleotides but also to the oligonucleotide p(dT) 8. All gp5-polynucleotide complexes give rise to intense linear dichroism spectra. These spectra are dominated by negative contributions from the bases, but also a small positive dichroism of the protein can be discerned. The spectra can be explained by polynucleotide structures, which are the same in all complexes. The base orientations are characterized by a substantial inclination and propellor twist. The number of possible combinations of inclination and propeller twist values, which are in agreement with the linear dichroism results, is rather limited. The base orientations with respect to the complex axis are essentially different from those in the complex with the single-stranded DNA-binding protein gp32 of bacteriophage T4.

Structure calculations for single-stranded DNA complexed with the single-stranded DNA binding protein GP32 of bacteriophage T4: a remarkable DNA structure

In this study it is established by calculation which regular conformations single-stranded DNA and RNA can adopt in the complex with the single-stranded DNA binding protein GP32 of bacteriophage T4. In order to do so, information from previous experiments about base orientations and the length and diameter of the complexes is used together with knowledge about bond lengths and valence angles between chemical bonds. It turns out that there is only a limited set of similar conformations which are in agreement with experimental data. The arrangement of neighboring bases is such that there is ample space for aromatic residues of the protein to partly intercalate between the bases, which is in agreement with a previously proposed model for the binding domain of the protein [Prigodich, R. V., Shamoo, Y., Williams, K. R., Chase, J. W., Konigsberg, W. H., & Coleman, J. E. (1986) Biochemistry 25, 3666-3671]. Both C2'endo and C3'endo sugar conformations lead to calculated DNA conformations that are consistent with experimental data. The orientation of the O2' atoms of the sugars in RNA can explain why the binding affinity of GP32 for polyribonucleotides is lower than for polydeoxyribonucleotides.

Alignment and merging of electron microscope images of frozen hydrated crystals of the T4 DNA helix destabilizing protein gp32*I

Low dose cryoelectron microscopy has been used to record images and electron diffraction patterns of frozen hydrated crystals of the single-stranded DNA binding protein gp32*I. Fourier transforms from 13 image areas, corresponding to approximately 40,000 unit cells, were aligned by a minimal phase residual search and merged by vector addition in reciprocal space. Phases from the resulting composite transform were combined with amplitudes from electron diffraction patterns to reconstruct the projected mass density of the gp32*I crystal at 8.4 A resolution.