Control of Helicase Loading in the Coupled DNA Replication and Recombination Systems of Bacteriophage T4

Abstract

Background: Helicase loading protein Gp59 coordinates recombination-dependent DNA replication in bacteriophage T4. Results: A DNA binding-deficient mutant, Gp59-I87A, loads helicase normally onto ssDNA covered with the ssDNA-binding protein, Gp32. Conclusion: Gp59-Gp32 interactions control helicase loading onto D-loops during recombination-dependent replication. Significance: Helicase loading proteins work in concert with single-stranded DNA binding proteins to control the location and timing of helicase assembly in DNA replication, recombination, and repair.

In vitro reconstitution of DNA replication initiated by genetic recombination: a T4 bacteriophage model for a type of DNA synthesis important for all cells

“…Because each protein binds cooperatively to DNA, with a gp32 monomer preferring to bind next to a previously bound gp32 molecule and a UvsX monomer preferring to bind next to a bound UvsX molecule, long alternating clusters of each type of protein are observed on a single–stranded DNA molecule (Griffith and Formosa, 1985). As others have studied in detail (Jones et al , 2001; Branagan et al , 2012, 2014), gp59 binds both to gp32 and to gp41, and it can act as a mediator to load gp41 onto patches of DNA covered with gp32. The results in Figure 1C demonstrate that this loading also occurs efficiently in the presence of excess UvsX protein.…”

“…The structures of both the gp59 and UvsY proteins have been determined by x-ray crystallography (Mueser et al , 2000; Gajewski et al , 2016), and their detailed modes of action have been analyzed by physicochemical techniques (Bleuit et al , 2001; Zhang et al , 2005; Liu et al , 2006, 2013; Xu et al , 2010; Branagan et al , 2012, 2014; Benkovic and Spiering, 2017). These small proteins readily oligomerize: gp59 to form hexamers and UvsY to form heptamers.…”

“…The addition of these two proteins had no effect on the recombination-dependent DNA synthesis reactions previously reported (Formosa and Alberts, 1986). Subsequently, the T4 gene 59 mediator protein (gp59) was discovered and characterized (Yonesaki, 1984; Barry and Alberts, 1994; Morrical et al , 1994, 1996); gp59 binds to both gp41 and gp32, and it thereby promotes the assembly of the T4 primosome on DNA (Jones et al ., 2001; Ma et al , 2004; Delagoutte and von Hippel, 2005; Branagan et al 2014; Benkovic and Spiering, 2017). The gp59 mediator (also termed a helicase-­assembly protein) is required for efficient recombination-dependent DNA replication inside a T4 bacteriophage-infected cell (Shah, 1976), and in this report we reexamine the properties of the above in vitro DNA replication system with this protein present.…”

Regulation of the bacteriophage T4 Dda helicase by Gp32 single-stranded DNA–binding protein

“…For all substrates, rates of unwinding were negligible in the presence of Gp32 alone or Gp32-B alone (data not shown). The forked substrates examined in Figure 4C–F are stable in the presence of Gp32 (Jones et al, 2004; Branagan et al, 2012, 2014), due to the fact that Gp32 is kinetically blocked from melting native duplex DNA (Jensen et al, 1976). With all substrates, the observed rates of Dda-catalyzed DNA unwinding decreased in the presence of Gp32, and, in all substrates except the simple 12:24 tailed duplex (Fig.…”

The Many Roles of Binding Cooperativity in the Control of DNA Replication

“…This binding saturation protects the ssDNA templates from cleavage by nucleases, prevents the formation of local secondary structure, and (at least for gp32) extends the ssDNA lattice to unstack neighboring bases, thus providing an optimal ssDNA template substrate for the T4 DNA polymerases (4). Gp32 also forms heterodimers at the replication fork with gp59 (the helicase-loading protein), likely speeding helicase loading by helping to displace the gp32 molecules bound directly at the replication fork junction that partially occlude the helicase-loading site, as well as facilitating recombination restart events at double-stranded DNA strand breaks and other sites of DNA damage (5). These elements of cooperativity must persist for the equivalent interactions in higher organisms as well, but their structural manifestations are clearly more complex and likely ''buried'' within their more complicated binding and interaction modes (Fig.…”