Biochemical and Electron Microscopic Image Analysis of the Hexameric E1 Helicase

Abstract

DNA replication initiator proteins bind site specifically to origin sites and in most cases participate in the early steps of unwinding the duplex. The papillomavirus preinitiation complex that assembles on the origin of replication is composed of proteins E1 and the activator protein E2. E2 is an ancillary factor that increases the affinity of E1 for the ori site through cooperative binding. Here we show that duplex DNA affects E1 (in the absence of E2) to assemble into an active hexameric structure. As a 10-base oligonucleotide can also induce this oligomerization, it seems likely that DNA binding allosterically induces a conformation that enhances hexamers. E1 assembles as a bi-lobed, presumably double hexameric structure on duplex DNA and can initiate bi-directional unwinding from an ori site. The DNA takes an apparent straight path through the double hexamers. Image analysis of E1 hexameric rings shows that the structures are heterogeneous and have either a 6-or 3-fold symmetry. The rings are about 40 -50 Å thick and 125 Å in diameter. The density of the central cavity appears to be a variable and we speculate that a plugged center may represent a conformational flexibility of a subdomain of the monomer, to date unreported for other hexameric helicases.The synthesis of duplex DNA is a complex enzymatic process that requires the coordination of large numbers of proteins. The mechanisms are elaborate in part because the enzymes that use the complementary template strand as a guide for nucleotide incorporation catalyze this synthesis only in the 5Ј to 3Ј direction. Given the antiparallel nature of the duplex this usually requires that two synthetic enzymes move in opposite polarities on the two strands. Synthesis of the so-called lagging strand is discontinuous and requires the cyclical association of the enzyme, while synthesis of the other strand is continuous. Nevertheless, in many prokaryote replication systems it is clear that coordination of these enzymes is achieved and maintained by a dimeric polymerase that creates a looped DNA structure in the lagging strand. This loop is mediated by multiple protein-protein interactions across the growing fork (Ref. 1, and references therein). Helicases are enzymes that can catalyze the unwinding of the template strands ahead of the fork, thus allowing for new complementary strand DNA synthesis. They were initially discovered as ancillary factors required for synthesis, but recently this view of the helicase activity has been characterized as "naive" or at least incomplete (2). Compelling evidence has been presented demonstrating that the helicase is an integral member of a large protein complex that serves as a molecular motor or pump for the replication apparatus empowering the polymerase and increasing the rate of DNA polymerase synthesis (3). The Escherichia coli dnaB helicase also plays a critical function in establishing the asymmetry at the growing fork. The helicase tracks on the lagging strand template but through interactions it holds the leading strand DN...

ATP-Dependent Minor Groove Recognition of TA Base Pairs Is Required for Template Melting by the E1 Initiator Protein

“…4B). The requirement for template melting indicates that formation of the DH helicase, similar to a single E1 hexamer, depends on ssDNA for its formation (12,30). Recent structural data demonstrate that the hexamer of the E1 helicase domain encircles ssDNA, and this is likely also the case for the DH (10).…”

On helicases and other motor proteins

“…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.…”

Substrate Requirements for Duplex DNA Translocation by the Eukaryal and Archaeal Minichromosome Maintenance Helicases

“…These proteins, however, also bind dsDNA but with much lower affinity (1,2,5,6). To date, high affinity dsDNA binding has been observed only for the viral encoded helicases, simian virus 40 large-T antigen (7) and the E1 protein from papilloma viruses (8). These proteins, in addition to their role as viral DNA replicative helicases, also bind to viral origins of replication acting as origin recognition proteins needed for the initiation of DNA replication.…”