Although relatively simple, the core activities of the T4 recombination system are highly conserved. Three core protein components are required for T4 presynaptic filament assembly and for DNA strand exchange under physiological conditions: UvsX, the phage recombinase (orthologous to bacterial RecA and eukaryotic Rad51); Gp32, the phage ssDNA-binding protein (equivalent to bacterial SSB and eukaryotic RPA); and UvsY, the phage recombination mediator protein (equivalent to bacterial RecOR, eukaryotic Rad52, Brca2, and others) [
4,
5]. The DNA binding properties of UvsX, Gp32, and UvsY are presented below in context with their physical and enzymatic properties.
UvsX recombinase
UvsX protein (44 kDa) is a member of the RecA/Rad51 recombinase family and shares 28% sequence identity and 51% sequence similarity with the catalytic core domain of
E. coli RecA [
12]. UvsX catalyzes DNA strand exchange reactions that play central roles in T4 HR, RDR, and HDR pathways [
4,
6]. UvsX binds sequence-non-specifically to both ssDNA and dsDNA and can bind to both lattices simultaneously via two different binding sites (Maher, R.L. and S.W. Morrical: Coordinated binding of ssDNA and dsDNA substrates by UvsX recombinase and its regulation by ATP, unpublished). UvsX has higher affinity for dsDNA in the absence of other factors, but simultaneous ssDNA binding lowers UvsX-dsDNA binding affinity unless the duplex sequence is homologous to the bound ssDNA (Maher, R.L. and S.W. Morrical: Coordinated binding of ssDNA and dsDNA substrates by UvsX recombinase and its regulation by ATP, unpublished). At the same time, UvsX-ssDNA interactions are selectively stabilized by nucleoside triphosphates ATP, dATP, or their non-hydrolyzable analogs, and by UvsY protein [
13,
14]. These combined factors help to target UvsX filament assembly onto recombinagenic ssDNA even in the presence of excess dsDNA as would normally be found in the T4-infected cell. Binding of UvsX to ssDNA, not dsDNA, specifically activates catalysis by UvsX including ATPase and DNA strand exchange activities.
Quantitative binding studies established the intrinsic ssDNA-binding parameters of UvsX [
13]. Its average binding site size on ssDNA is 4 nucleotide residues per protomer. UvsX exhibits moderate affinity and cooperativity for ssDNA with
Kobs =
K ω ≈ 10
6 M
-1 at physiological ionic strength, where the cooperativity parameter ω ≈ 100 [
13]. The observed cooperativity of UvsX is consistent with the formation of long filaments on ssDNA at high binding density.
The ATPase activity of UvsX is strongly ssDNA-dependent under normal solution conditions [
15], although very high salt concentrations can also stimulate ATP hydrolysis by UvsX in the absence of ssDNA. Double-stranded DNA does not activate UvsX ATPase activity. UvsX ATPase activity is also highly unusual in that it generates both ADP and AMP as products [
15,
16]. The two products appear to be generated independently by two different classes of active sites within UvsX-ssDNA presynaptic filaments, as indicated by results of steady-state kinetics studies [
16]. These sites have different
Km and
kcat/
Km values for the ATP and ssDNA substrates. One type of active site appears to produce ADP exclusively, while the other appears to generate AMP via a sequential mechanism (ATP → ADP → AMP) without releasing the ADP intermediate from the active site [
16]. Thus UvsX presynaptic filaments exhibit active site asymmetry (Figure
2). This asymmetry may be important for UvsX-catalyzed DNA strand exchange reactions, since increases in ADP/AMP product ratio observed in UvsX site-directed mutants correlate inversely with strand exchange activity [
16]. Active site asymmetry may be a general property of presynaptic filaments in many species, since evidence exists for two classes of active sites in filaments of
E. coli RecA and
S. cerevisiae Rad51 recombinases [
17,
18].
UvsX-ssDNA filaments rapidly search for homology in dsDNA substrates, leading to efficient homologous pairing and strand exchange. ATP binding (not hydrolysis) is required for homologous pairing, however ATP hydrolysis is needed to drive extensive polar (5' → 3') branch migration during strand exchange [
19‐
21]. There is a strong requirement for Gp32 to stimulate UvsX-catalyzed strand exchange at normal concentrations of the recombinase [
15,
22,
23]. In vitro, this Gp32 requirement can be circumvented by raising the UvsX concentration to super-saturating levels with respect to ssDNA binding sites. Stimulation of strand exchange by Gp32 requires the correct order of protein addition: Adding Gp32 to ssDNA prior to the addition of UvsX typically inhibits strand exchange. This ssDNA-binding protein/recombinase order of addition effect is a characteristic of all well-characterized recombination systems [
24], and is reflective of the competition between the two proteins for binding sites on ssDNA. Similar inhibition of UvsX-catalyzed strand exchange is seen at high concentrations of Gp32 and/or at elevated salt concentrations, i.e. conditions that favor Gp32-ssDNA over UvsX-ssDNA interactions. Under conditions such as these there is an absolute requirement for the UvsY recombination mediator protein for strand exchange reactions in vitro [
23,
25]. This mimics the in vivo situation in which T4 recombination transactions are equally dependent on UvsX and UvsY [
26‐
28].
Branched networks of single- and double-stranded DNA are the major products of UvsX-catalyzed DNA strand exchange, indicating that each DNA substrate molecule participates in many homologous pairing events [
15,
29]. One plausible explanation for this behavior is that UvsX appears to catalyze homologous pairing much more rapidly than branch migration. Therefore it is possible for different regions of one long ssDNA substrate to pair with homologous regions of different dsDNA substrates before any of the resulting D-loop intermediates can be completely extended into heteroduplex DNA. Rapid homologous pairing by UvsX may be an evolutionary adaptation for efficiently capturing 3' ssDNA tails and using them to prime recombination-dependent replication. Furthermore, branch migration appears to be dependent on T4-encoded DNA helicases, as we discuss in a later section.
Gp32 ssDNA-binding protein
Gp32 (34 kDa) is the prototype ssDNA-binding protein and a key component of the T4 replisome. Gp32 also plays important roles in homologous recombination and DNA repair. The biochemical properties of Gp32 have been thoroughly characterized [
30‐
45], and the atomic structure of its central DNA-binding domain (DBD) has been solved [
32]. The DBD contains an oligonucleotide/oligosaccharide-binding (OB)-fold motif plus a structural Zn
++ atom. An N-terminal domain (so-called
basic or "B-domain") is required for self-association and cooperativity, whereas a C-terminal domain (so-called
acidic or "A-domain") is the site for protein-protein interactions with various recombination and replication enzymes including UvsX and UvsY.
Gp32 binds sequence-non-specifically to polynucleotides, with the highest observed affinity for ssDNA (Kobs ≈ 109 M-1 at physiological ionic strength), moderate affinity for single-stranded RNA, and very low affinity for dsDNA. The binding site size of Gp32 on ssDNA is approximately 7 nucleotide residues. Binding to ssDNA is highly cooperative (ω ≈ 1000), meaning that Gp32 exists almost exclusively in clusters or long filaments on ssDNA at protein concentrations normally encountered in in vitro DNA strand exchange assays as well as in vivo.
Gp32 affects both pre- and post-synaptic steps of UvsX-catalyzed DNA strand exchange reactions [
15,
22,
23,
25,
46,
47]. An important function of Gp32 in presynapsis is to denature secondary structure in the ssDNA substrate, which
eventually allows UvsX to saturate the ssDNA by forming long presynaptic filaments. Paradoxically, the immediate effect of Gp32 on UvsX-ssDNA filament formation is negative under physiological conditions, because Gp32 competes effectively with UvsX for binding sites [
13]. Overcoming Gp32 inhibition requires either pre-incubation of UvsX with ssDNA in the presence of ATP (the previously mentioned order of addition effect), or the inclusion of UvsY in reaction mixtures (see below) [
4,
24]. Gp32 has also been shown to play a post-synaptic role in strand exchange, stimulating the reaction by sequestering the outgoing ssDNA strand that is displaced during D-loop formation and subsequent branch migration [
47].
UvsY is the prototype recombination mediator protein or RMP [
24]. By definition, RMPs are proteins that load recombinases of the RecA/Rad51 family onto ssDNA molecules that are pre-saturated with cognate ssDNA-binding protein. UvsY is absolutely required for UvsX-catalyzed DNA strand exchange in the presence of Gp32 under physiological or high-salt conditions [
22,
48,
49]. In vivo, UvsY is also absolutely required for UvsX-dependent recombination since mutations knocking out either gene product have equivalent recombination-deficient phenotypes including the small-plaque phenotype associated with defective RDR [
26‐
28]. UvsY is the only member of the core T4 recombination machinery that forms a discreet oligomeric structure: It exists as a stable hexamer of identical 15.8 kDa subunits in solution, and binds to ssDNA in this form [
50].
UvsY binds to both ssDNA and dsDNA, but has a much higher affinity for the former under relaxed DNA conditions [
51]. The preference of UvsY for ssDNA may be an important factor in directing UvsX filament assembly onto ssDNA in the presence of excess dsDNA, since UvsX itself has a relatively high affinity for non-homologous dsDNA (Maher, R.L. and S.W. Morrical: Coordinated binding of ssDNA and dsDNA substrates by UvsX recombinase and its regulation by ATP, unpublished). UvsY has a binding site size on ssDNA of 4 nucleotide residues per protomer, or 24 nucleotide residues per hexamer [
52]. The protomeric binding site sizes of UvsY and UvsX are identical. UvsY binds to ssDNA with high affinity (
K-
obs ≈ 10
7 M
-1 at physiological ionic strength), but with little or no cooperativity (ω ≈ 1). Therefore UvsY has higher
intrinsic affinity, but lower cooperativity, for ssDNA than either UvsX or Gp32 under conditions that are relevant for strand exchange reactions in vitro and in vivo. UvsY-ssDNA interactions are weakened by mutations at residues Lys-58 and Arg-60, which form part of a conserved LKARLDY motif (so-called 'KARL' motif) found in the N-terminal domain of UvsY, which is thought to comprise part of its DNA binding surface [
14,
48,
51,
53,
54]. The KARL motif is also found in certain DNA helicases, however no helicase activity has ever been associated with UvsY, which lacks a motor domain. The C-terminal domain of UvsY is essential for hexamerization. Deletion of this domain drastically reduces the affinity of UvsY-ssDNA interactions, demonstrating the importance of UvsY hexamers as the relevant ssDNA-binding unit [
55].
Several lines of evidence indicate that UvsY hexamers have the ability to wrap ssDNA strands around themselves, and that wrapping is responsible for the high affinity of UvsY-ssDNA interactions. Evidence includes the observation that a C-terminally deleted, monomeric form of UvsY has 10
4-fold lower affinity for ssDNA than wild-type [
55]. The wrapping hypothesis is supported by the finding that mutiple subunits within each UvsY hexamer are in contact with ssDNA [
51]. Other evidence comes from results of single-molecule DNA stretching studies, which showed that the ssDNA that is created by the treatment of individual stretched dsDNA molecules with glyoxal is strongly wrapped by UvsY [
54]. Wrapping of ssDNA occurs at low stretching forces where the DNA is relatively relaxed. At high stretching forces, where the DNA is under tension, wrapping is suppressed. The tension-dependent suppression of wrapping leads to the loss of preferential binding to ssDNA as shown by the fact that UvsY binds tighter to stretched dsDNA than to stretched ssDNA [
54]. This contrasts with the observation that UvsY has ~1000-fold higher affinity for ssDNA than for dsDNA under relaxed conditions [
51]. Therefore high-affinity binding of UvsY to ssDNA requires wrapping, which also imposes a preference for binding to ssDNA over dsDNA. Presumably UvsY cannot wrap dsDNA because its persistence length is much higher than that of ssDNA [
56]. The surprising observation that UvsY binds tightly to stretched dsDNA could have important implications for presynaptic filament assembly. The binding of Gp32 to ssDNA creates an extended or "stiff" DNA conformation that might be recognized by UvsY in an unwrapped mode similar to its interaction with stretched dsDNA. Converting this extended ssDNA structure into a wrapped one might be an important step in the recruitment of UvsX recombinase, as we discuss in a later section.
UvsY is absolutely required for UvsX-catalyzed DNA strand exchange assays performed under physiological conditions of Gp32 and salt [
4,
24], consistent with the co-dependency of recombination on UvsX and UvsY in vivo [
26‐
28]. In vitro, UvsY lowers the critical concentration of UvsX for RDR and other recombination reactions [
46,
57]. UvsY stimulates the ssDNA-dependent ATPase activity of UvsX, possibly by acting as a nucleotide exchange factor for the recombinase [
58]. The greatest stimulation of ATPase activity is seen when UvsY and Gp32 act together synergistically on the reaction [
23,
49]. UvsY stimulates the catalytic activities of UvsX mainly by promoting presynaptic filament assembly. The mechanism of UvsY's recombination mediator activity will be explored in greater detail below.