Physical biochemical techniques are used to establish the structure, subunit stoichiometry, and assembly pathway of the primosome complex of the bacteriophage T4 DNA replication system. Analytical ultracentrifugation and fluorescence anisotropy methods show that the functional T4 primosome consists of six gp41 helicase subunits that assemble into a hexagon, driven by the binding of six NTPs (or six nonhydrolyzable GTPγS analogues) that are located at and stabilize the intersubunit interfaces, together with a single tightly bound gp61 primase subunit. Assembling the components of the primosome onto a model DNA replication fork is a multistep process, but equilibrium cannot be reached along all mixing pathways. Producing a functional complex requires that the helicase hexamer be assembled in the presence of the DNA replication fork construct prior to the addition of the primase to avoid the formation of metastable DNA-protein aggregates. The gp41 helicase hexamer binds weakly to fork DNA in the absence of primase, but forms a much more stable primosome complex that expresses full and functional helicase (and primase) activities when bound to a gp61 primase subunit at a helicase:primase subunit ratio of 6∶1. The presence of additional primase subunits does not change the molecular mass or helicase activity of the primosome, but significantly inhibits its primase activity. We develop both an assembly pathway and a minimal mechanistic model for the structure and function of the T4 primosome that are likely to be relevant to the assembly and function of the replication primosome subassemblies of higher organisms as well.DNA-protein complexes | macromolecular machines | duplex DNA unwinding | replication complex assembly T he DNA replication system of bacteriophage T4 contains eight different types of protein subunits, several present in multiple copies. Subsets of these components form three stable and functional protein complexes that can be assembled onto a model DNA replication fork in vitro to form an integrated T4 DNA replication complex that is capable of unwinding the parental DNA duplex and synthesizing new viral DNA with essentially in vivo rates and fidelity (1, 2). These replication subassemblies are: (i) the leading-and lagging-strand replication polymerases that catalyze the template-directed copying of the two parental-strands of the DNA genome at each cell division; (ii) the clamp-clamp loader complex that controls the processivity of the replication process by linking the polymerases to their respective template strands and also regulates the release and recycling of the lagging strand polymerase following the completion of each Okazaki fragment; and (iii) the helicase-primase (primosome) complex that unwinds the double-stranded genome ahead of the replication fork in its capacity as a helicase, while also performing template-directed synthesis of the RNA primers that reinitiate discontinuous lagging-strand DNA synthesis after each Okazaki fragment has been completed.The subunit components and stoichio...

A sensitive two-color electrophoretic mobility shift assay for detecting both nucleic acids and protein in gels DNA-binding proteins are key to the regulation and control of gene expression, replication and recombination. The electrophoretic mobility shift assay (or gel shift assay) is considered an essential tool in modern molecular biology for the study of proteinnucleic acid interactions. As typically implemented, however, the technique suffers from a number of shortcomings, including the handling of hazardous 32 P-labeled DNA probes, and difficulty in quantifying the amount of DNA and especially the amount of protein in the gel. A new detection method for mobility-shift assays is described that represents a significant improvement over existing techniques. The assay is fast, simple, does not require the use of radioisotopes and allows independent quantitative determination of: (i) free nucleic acid, (ii) bound nucleic acid, (iii) bound protein, and (iv) free protein. Nucleic acids are detected with SYBR  Green EMSA dye, while proteins are subsequently detected with SYPRO  Ruby EMSA dye. All fluorescence staining steps are performed after the entire gel-shift experiment is completed, so there is no need to prelabel either the DNA or the protein and no possibility of the fluorescent reagents interfering with the protein-nucleic acid interactions. The ability to independently quantify each molecular species allows more rigorous data analysis methods to be applied, especially with respect to the mass of protein bound per nucleic acid.

One primase (gp61) and six helicase (gp41) subunits interact to form the bacteriophage T4-coded primosome at the DNA replication fork. In order to map some of the detailed interactions of the primase within the primosome, we have constructed and characterized variants of the gp61 primase that carry kinase tags at either the N or the C terminus of the polypeptide chain. These tagged gp61 constructs have been probed using several analytical methods. Proteolytic digestion and protein kinase protection experiments show that specific interactions with single-stranded DNA and the T4 helicase hexamer significantly protect both the N-and the Cterminal regions of the T4 primase polypeptide chain against modification by these procedures and that this protection becomes more pronounced when the primase is assembled within the complete ternary primosome complex. Additional discrete sites of both protection and apparent hypersensitivity along the gp61 polypeptide chain have also been mapped by proteolytic footprinting reactions for the binary helicase-primase complex and in the three component primosome. These studies provide a detailed map of a number of gp61 contact positions within the primosome and reveal interactions that may be important in the structure and function of this central component of the T4 DNA replication complex.Most known DNA replication complexes, including the bacteriophage T4-coded system, are organized into subassemblies of proteins that perform equivalent functions within the moving replication fork. Since the T4 complex is probably the simplest replication system that includes separate processivity clamp and clamp loader and primosome subassemblies, the T4 system has often served as a useful paradigm to elucidate and model some of the mechanistically important interactions that also occur within the replication complexes of higher organisms.The central replication machinery of bacteriophage T4 consists of seven phage-encoded proteins that work together with the DNA of the replication fork to form the minimal complex that permits efficient leading and lagging DNA synthesis and replication fork movement (1). These protein components are organized into three functional subassemblies within the replication complex. These are (i) the DNA-dependent DNA replication polymerase of T4 (gp43), which contains both synthesis and exonuclease (3Ј 3 5Ј) editing domains and works together with the T4-coded single-stranded DNA binding protein (gp32) to catalyze DNA synthesis on both strands of the replication fork; (ii) the T4-coded polymerase accessory proteins (gp44/62 and gp45), which form the processivity clamp (gp45) and ATPdependent clamp-loading (gp44/62) machinery of the T4 system; and (iii) the T4 coded helicase (gp41) and primase (gp61) components, which interact to form a primosome subassembly that contains an ATP-dependent helicase (gp41) that "opens" the template DNA ahead of the replication fork and a primase (gp61) that catalyzes the template-directed polymerization of the RNA primers that initiate th...

The novel method showed satisfactory assay performance in addition to drastically reduced analysis times and improved ease of use as compared to other methods. Clinical utility of HDL 2b was demonstrated supporting the findings of previous studies.

Protein-DNA and protein-protein interactions play essential roles in many biologic processes such as transcription, replication, recombination, and DNA repair. One of the most popular approaches to investigate specific protein-nucleic acid interactions is the electrophoretic mobility shift assay (EMSA). We have developed a new nonradioactive method to detect both nucleotides and protein in EMSA. Nucleic acids are detected with SYBR Green EMSA dye, while proteins are subsequently detected with SYPRO Ruby EMSA dye. All fluorescent staining steps are performed after the entire gel-shift experiment is completed, so there is no need to prelabel either the nucleic acids or the protein. The two-color fluorescence dye staining procedure is fast, simple, and allows independent quantitative determination of either free or bound nucleic acids and protein. The interactions between lac repressor-operator, and among the T4 primase-helicase, primase-DNA, helicase-DNA, and within T4 [ssDNA-primase-helicase6] primosome, were used to demonstrate the advantages of this two-color fluorescence detection EMSA method.