Bacteriophage T7 helicase/primase proteins form rings around single-stranded DNA that suggest a general structure for hexameric helicases.

Abstract

Most helicases studied to date have been characterized as oligomeric, but the relation between their structure and function has not been understood. The bacteriophage T7 gene 4 helicase/primase proteins act in T7 DNA replication. We have used electron microscopy, threedimensional reconstruction, and protein crosslinking to demonstrate that both proteins form hexameric rings around single-stranded DNA. Each subunit has two lobes, so the hexamer appears to be two-tiered, with a small ring stacked on a large ring. The single-stranded DNA passes through the central hole of the hexamer, and the data exclude substantial wrapping of the DNA about or within the protein ring. Further, the hexamer binds DNA with a defined polarity as the smaller ring of the hexamer points toward the 5' end of the DNA. The similarity in three-dimensional structure of the T7 gene 4 proteins to that of the Escherichia coli RuvB helicase suggests that polar rings assembled around DNA may be a general feature of numerous hexameric helicases involved in DNA replication, transcription, recombination, and repair.DNA helicases are ubiquitous proteins that unwind doublestranded DNA (dsDNA) into single-stranded DNA (ssDNA), using energy from NTP hydrolysis (1). Interestingly, most of the helicases that have been studied to date self-assemble into either dimers or hexamers (2, 3). The significance of oligomerization in helicases is unclear, as the mechanism of DNA unwinding is not understood. Bacteriophage T7 gene 4 proteins provide the helicase and primase functions for T7 DNA replication and both proteins have been shown to form stable hexamers in the presence of Mg2+ and thymidine 5'-[I,3y-methylene]triphosphate (dTTP[3,), a nonhydrolyzable analog of dTTP (4). All data suggest that the hexamer is the active form of the protein involved in DNA unwinding, since hexamer formation is required for DNA binding, and substitution of a few of the hexameric subunits with inactive subunits inhibits both the DNA-dependent dTTPase and the helicase activities of the protein (5). We report here the structures of T7 gene 4A' and 4B proteins and their mode of DNA binding, using the techniques of electron microscopy, threedimensional reconstruction, and protein crosslinking. MATERIALS AND METHODSPreparation of Gene 4 Protein-DNA Complexes. T7 gene 4A' and 4B proteins were prepared as described (6). Preparations in the absence of DNA involved incubations of the protein (0.7 ,uM) for 10 min at 37°C in 25 mM triethanolamine buffer (pH 7.2) with 2 mM magnesium acetate and 1.3 mM dTTP[P3,y-CH2]. Preparations with ssDNA were similar, ex-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3869cept that the protein concentration was 1.5 ,uM and the DNA concentration was 0.6 nM.Synthesis of a-32P-Labeled-ssDNA. Radiolabeled linear ssDNA was prepared by elongating a 30-mer oligodeoxynucleotide with terminal deox...

On helicases and other motor proteins

“…Most, perhaps all, hexameric helicases operate by this model based upon their ability to pass over a bulky substituent when it is present upon one strand but not the other. Bacteriophage T7gp4B was shown by EM to form hexameric ring structures on M13 DNA [11] and to cleanly unwind with 5 0 to 3 0 polarity if a bulky substrate was placed on the 5 0 strand [28]. For strand displacement assays that incorporate a labeled single-stranded oligonucleotide annealed to M13 DNA, the closed circular M13 DNA itself constitutes the bulky substituent.…”

“…Superfamily 1 (SF1) and superfamily 2 (SF2) helicases are very prevalent, generally monomeric, and participate in several diverse DNA and RNA manipulations. The other helicase superfamilies form hexameric rings (reviewed in [4]), as demonstrated by biochemistry [5][6][7][8] and electron microscopy studies [9][10][11][12][13][14][15][16][17], and often participate at the replication fork. All of these helicases bind and hydrolyze NTP at the interface between two recA-like domains.…”

DNA Looping Mediates Nucleosome Transfer

“…8. For the leading strand replication gel experiments, the initial replication fork consisted of a trunk of a 298 bp dsDNA containing the 601 nucleosome-positioning element, a 30-nucleotide flap of ssDNA to facilitate T7 helicase loading to the replication fork 29,31,56 , and a 1,189 bp dsDNA leading strand.…”

Communication between subunits critical to DNA binding by hexameric helicase of bacteriophage T7

“…Electron microscopy and x-ray crystallography illustrated that the protein forms a closed ring-shaped oligomeric structure composed of six or seven subunits (4)(5)(6)(7). Biochemical studies have identified regions critical to oligomerization of the protein, including a linker region that connects the C-terminal helicase domain of the protein to the N-terminal primase domain (8,9).…”