Mediator proteins orchestrate enzyme-ssDNA assembly during T4 recombination-dependent DNA replication and repair

Abstract

Studies of recombination-dependent replication (RDR) in the T4 system have revealed the critical roles played by mediator proteins in the timely and productive loading of specific enzymes onto single-stranded DNA (ssDNA) during phage RDR processes. The T4 recombination mediator protein, uvsY, is necessary for the proper assembly of the T4 presynaptic filament (uvsX recombinase cooperatively bound to ssDNA), leading to the recombination-primed initiation of leading strand DNA synthesis. In the lagging strand synthesis component of RDR, replication mediator protein gp59 is required for the assembly of gp41, the DNA helicase component of the T4 primosome, onto lagging strand ssDNA. Together, uvsY and gp59 mediate the productive coupling of homologous recombination events to the initiation of T4 RDR. UvsY promotes presynaptic filament formation on 3' ssDNA-tailed chromosomes, the physiological primers for T4 RDR, and recent results suggest that uvsY also may serve as a coupling factor between presynapsis and the nucleolytic resection of double-stranded DNA ends. Other results indicate that uvsY stabilizes uvsX bound to the invading strand, effectively preventing primosome assembly there. Instead, gp59 directs primosome assembly to the displaced strand of the D loop/replication fork. This partitioning mechanism enforced by the T4 recombination/replication mediator proteins guards against antirecombination activity of the helicase component and ensures that recombination intermediates formed by uvsX/uvsY will efficiently be converted into semiconservative DNA replication forks. Although the major mode of T4 RDR is semiconservative, we present biochemical evidence that a conservative "bubble migration" mode of RDR could play a role in lesion bypass by the T4 replication machinery.

Figure 1

T4 in vitro system for RDR. Step 1: The 3′ end of linear ssDNA primer (blue) invades homologous dsDNA template (red) in reaction catalyzed by uvsX recombinase and stimulated by uvsY and gp32. Step 2: DNA polymerase holoenzyme uses primer terminus in D loop to initiate leading strand synthesis. Reaction requires a functional T4 DNA helicase (either dda or gp41/gp59). Branch migration of the trailing junction displaces the daughter strand from the template, leading to conservative (a.k.a. bubble migration) DNA synthesis. Step 3: Strand-specific assembly of functional primosome (gp41 helicase/gp61 primase) on displaced strand of D loop, a reaction requiring gp59. Lagging strand synthesis within the “bubble” reconstitutes a semiconservative replication fork, freezes branch migration and halts bubble migration synthesis.

Figure 2

(A) Biochemical model for uvsY-mediated assembly of the T4 presynaptic filament. Step 1: Hexameric uvsY protein binds to gp32-ssDNA complex and destabilizes gp32-ssDNA interactions. Step 2: UvsX recombinase is recruited to the uvsY-gp32-ssDNA intermediate and locally displaces gp32 to nucleate a filament. Step 3: UvsX-ssDNA filament assembly propagates in the 5′ → 3′ direction while displacing gp32. (B) Hypothetical model for presynapsis coupled to gp46/47-catalyzed resection of a DSB. Step 1: DSB is resected in 5′ → 3′ direction by gp46/47 exonuclease activity, generating a 3′ ssDNA tail. Gp46/47 simultaneously recruits uvsY protein, which in turn recruits uvsX. Step 2: Ongoing recruitment of uvsX by gp46/47 + uvsY leads to continuous presynaptic filament formation as the ssDNA tail is exposed by nuclease action, preparing the tail for immediate entry into RDR/double-strand break repair processes.

Figure 3

Biochemical model for gp59-mediated helicase assembly at T4 replication fork. Step 1: Nascent strand-displacing replication fork (DNA polymerase holoenzyme plus gp32). Cluster of gp32-gp59 complexes is incorporated at growing end of gp32 lagging-strand complex. Affinity of gp59 for fork DNA may help nucleate the cluster. Step 2: Gp59 recruits dimers of gp41 helicase to the cluster and promotes displacement of gp32. Step 3: Gp59 stimulates ATP binding by gp41, triggering ring-hexamer formation by the helicase. Gp41-gp59 complex translocates with the replication fork, ready to recruit primase.

Figure 4

Enzyme partitioning model for strand-specific priming of Okazaki fragments during T4 recombination-dependent replication. Step 1: Gp32 sequesters the displaced strand of the D loop after uvsX-catalyzed invasion of the primer strand. A cluster of gp32-gp59 complexes forms on the displaced strand as shown in Fig. 3. The invading strand ssDNA 5′ of the trailing junction remains sequestered by a uvsY-stabilized uvsX-ssDNA presynaptic filament. Gp41 helicase assembly therefore is directed to the gp32/gp59-enriched D-loop ssDNA and is precluded from the invading strand. Step 2: Recruitment of primase by the gp41 helicase reconstitutes lagging strand synthesis and a normal semiconservative replication fork specifically from within the D-loop bubble.

Figure 5

Template switching assay for lesion bypass under conditions supporting bubble migration synthesis by the T4 RDR in vitro system. (A) DNA primer and template design for template switching assay. Damaged template contains a site-specific psoralen monoadduct on the (−) strand at position 6276 in M13mp19 dsDNA. Supercoiled, psoralenated DNA was synthesized as described (35), then linearized with SnaBI. Homologous rescue template is the short BalI–BglII restriction fragment of M13mp19 dsDNA, which overlaps the psoralenated site in the damaged template. The ssDNA primer is a 5′-[³²P]-100-mer complementary to the (−) strand of M13mp19 dsDNA at positions 4806–4905. This primer initiates RDR on the damaged template but not on the homologous rescue template. The dotted arrow denotes the replicative path needed to generate the switching product (see text). (B) Template switching assay and controls. Reactions (15 μl) at 37°C contained 20 mM Tris-acetate, pH 7.4, 10 mM magnesium acetate, 90 mM potassium acetate, 0.5 mM DTT, 50 μg/ml BSA, 10 μg/ml creatine phosphokinase, 10 mM creatine phosphate, 0.25 mM spermine, 0.15 mM spermidine, 3.8 μg/ml gp43, 25 μg/ml gp44/62, 8 μg/ml gp45, 85 μg/ml gp32, 4 μg/ml dda, 18 μg/ml gp41, 2 μg/ml gp59, 20 μg/ml uvsX, 5 μg/ml uvsY, 2 mM ATP, 1.75 mM GTP, 200 μM each dATP, dGTP, dCTP, and dTTP, 2 μM 5′-[³²P]-100-mer, 10 μM damaged template, and 10 μM homologous rescue template. (All DNA concentrations in nucleotides.) Some control reactions contained 10 μM heterologous pBR322 restriction fragments in place of homologous templates. Each reaction was initiated by the addition of DNAs and nucleotides, incubated for 10 min, then analyzed by alkaline agarose gel electrophoresis and autoradiography as described (4). Lane 1: Size marker for position of stall product. Lane 2: Complete template switching reaction containing primer, damaged template, and homologous rescue template. Lane 3: Control reaction containing XmnI-digested pBR322 dsDNA in place of damaged template. Lane 4: Control reaction containing XmnI-digested pBR322 dsDNA in place of homologous rescue template. (C) Lesion dependence of template switching. Lane 1: Complete reaction identical to lane 2 of B, except that a longer ssDNA primer was used (2,527-b HaeIII fragment of M13 ssDNA), and DNA synthesis products were visualized by incorporation of α-[³²P]-dTTP. Lane 2: Same as lane 1 except that the damaged template lacks psoralen.