Dual Functions of Single-stranded DNA-binding Protein in Helicase Loading at the Bacteriophage T4 DNA Replication Fork

Abstract

Semi-conservative DNA synthesis reactions catalyzed by the bacteriophage T4 DNA polymerase holoenzyme are initiated by a strand displacement mechanism requiring gp32, the T4 single-stranded DNA (ssDNA)-binding protein, to sequester the displaced strand. After initiation, DNA helicase acquisition by the nascent replication fork leads to a dramatic increase in the rate and processivity of leading strand DNA synthesis. In vitro studies have established that either of two T4-encoded DNA helicases, gp41 or dda, is capable of stimulating strand displacement synthesis. The acquisition of either helicase by the nascent replication fork is modulated by other protein components of the fork including gp32 and, in the case of the gp41 helicase, its mediator/ loading protein gp59. Here, we examine the relationships between gp32 and the gp41/gp59 and dda helicase systems, respectively, during T4 replication using altered forms of gp32 defective in either protein-protein or protein-ssDNA interactions. We show that optimal stimulation of DNA synthesis by gp41/gp59 helicase requires gp32-gp59 interactions and is strongly dependent on the stability of ssDNA binding by gp32. Fluorescence assays demonstrate that gp59 binds stoichiometrically to forked DNA molecules; however, gp59-forked DNA complexes are destabilized via protein-protein interactions with the C-terminal "A-domain" fragment of gp32. These and previously published results suggest a model in which a mobile gp59-gp32 cluster bound to lagging strand ssDNA is the target for gp41 helicase assembly. In contrast, stimulation of DNA synthesis by dda helicase requires direct gp32-dda protein-protein interactions and is relatively unaffected by mutations in gp32 that destabilize its ssDNA binding activity. The latter data support a model in which protein-protein interactions with gp32 maintain dda in a proper active state for translocation at the replication fork. The relationship between dda and gp32 proteins in T4 replication appears similar to the relationship observed between the UL9 helicase and ICP8 ssDNA-binding protein in herpesvirus replication.DNA helicase acquisition by a nascent DNA replication fork is essential for reconstituting rapid, processive DNA synthesis. This principle is illustrated dramatically by the bacteriophage T4 DNA replication system (1-2). T4 encodes two DNA helicases known to affect the movement and properties of DNA replication forks; the gene 41 protein (gp41) and the dda protein, respectively. gp41 is the essential replicative helicase of the T4 phage. This hexameric enzyme translocates processively in a 5Ј 3 3Ј direction on the displaced lagging strand of the dsDNA 1 template, thus enhancing the rate and processivity of leading strand DNA synthesis catalyzed by the T4 DNA polymerase holoenzyme (gp43, gp44/62, and gp45 proteins) (1-3). In addition, gp41 is an obligatory component of the T4 primosome (helicase/primase complex) and is, thus, essential for lagging strand DNA synthesis (4 -5). A second T4-encoded DNA helicase, dda protein, stimula...

Models for the Binary Complex of Bacteriophage T4 Gp59 Helicase Loading Protein

“…Our data agrees with earlier studies indicating that gp59 protein binds to fork DNA and recruits gp32 protein to DNA structures with at least 9 nucleotides on the lagging strand arm (5). Also in agreement is the effect of gp32 and gp32⌬B on the affinity of the gp59 protein for fork DNA (13,34). The relative weak level of incorporation of gp32⌬B protein into the gp59-DNA complex and the propensity of these ternary complexes to aggregate at higher concentrations precluded further analysis.…”

Templated Mutagenesis in Bacteriophage T4 Involving Imperfect Direct or Indirect Sequence Repeats

“…Gp32 binds to single-stranded DNA and helps, directly or indirectly, to load several replication proteins and to stabilize the replication fork. Although gp32 does not bind gp41, it does interact with gp59 which in turn loads gp41 (Ma et al 2004;Delagoutte and Von Hippel 2005;Xi et al 2005;Zhang et al 2005). Thus, when gp32 is partly dysfunctional, as in gp32mms, the primer terminus would more often be exposed, and/or the single-stranded portion of template would more often not be coated with gp32, increasing primerterminus melting and its mutagenic consequences; the allelic state of gene 41 would not matter very much, as observed (Tables 5 and 6).…”

Bacteriophage T4 Helicase Loader Protein gp59 Functions as Gatekeeper in Origin-dependent Replication in Vivo

“…In vitro replication assays using the synthetic R-loop substrate first suggested that gp59 blocks the progression of DNA polymerase in the absence of gp41 (16). This conclusion was confirmed by additional studies using either singly primed M13 DNA (extended by holoenzyme in the first stage of a two-stage reaction) or a nicked substrate that is replicated by a rolling circle mechanism (23,24). More recently, Xi et al (47) have presented a detailed study of gp59-mediated inhibition of T4 DNA polymerase, showing that the inhibition involves a direct protein-protein interaction (also see below).…”

“…How does gp41 load without gp59? The displaced strand of the origin R-loop presents a likely loading site for the initial fork, because gp41 can load onto single-stranded DNA in vitro in the absence of gp59 (17,19,23,24). However, the loading of gp41 for the retrograde fork in the absence of gp59 may be more difficult, because the lagging-strand template for that fork starts out duplex (see Fig.…”

“…In addition, gp59 has been shown to bind tightly to gp32, which might also help target gp59/41 to these initiating DNA structures (23, 24, 26 -28). The gp59-mediated assembly of gp41 onto gp32-coated single-stranded DNA most likely involves a ternary complex between these three proteins (23,24,29). Recent in vitro studies indicate that gp59 travels with the replication fork, perhaps playing a role in the coupling of leading-and lagging-strand synthesis (30,31).…”