Multiple C-terminal tails within a single E. coli SSB homotetramer coordinate DNA replication and repair

Abstract

Escherichia coli single-stranded DNA binding protein (SSB) plays essential roles in DNA replication, recombination and repair. SSB functions as a homotetramer with each subunit possessing a DNA binding domain (OB-fold) and an intrinsically disordered C-terminus, of which the last nine amino acids provide the site for interaction with at least a dozen other proteins that function in DNA metabolism. To examine how many C-termini are needed for SSB function, we engineered covalently linked forms of SSB that possess only one or two C-termini within a four-OB-fold "tetramer". Whereas E. coli expressing SSB with only two tails can survive, expression of a single-tailed SSB is dominant lethal. E. coli expressing only the two-tailed SSB recovers faster from exposure to DNA damaging agents but accumulates more mutations. A single-tailed SSB shows defects in coupled leading and lagging strand DNA replication and does not support replication restart in vitro. These deficiencies in vitro provide a plausible explanation for the lethality observed in vivo. These results indicate that a single SSB tetramer must interact simultaneously with multiple protein partners during some essential roles in genome maintenance.

Keywords: DNA binding; DNA repair; DNA replication; EDTA; FRET; PBS; SIP; SSB; SSB interacting protein; ethylenediaminetetraacetic acid; fluorescence resonance energy transfer; phosphate-buffered saline; single stranded DNA binding protein; single-stranded DNA; single-stranded DNA binding protein; ssDNA; wild type; wt.

Figure 1

Design of covantly linked SSB proteins. A) Schematic of the linker design used to generate the linked SSB dimer (SSB-LD-Drl) and the linked SSB tetramer (SSB-LT-Drl) resulting in two and one C-terminal tail per 4-OB folds respectively. B) Superimposition of one Dr-SSB monomer containing two OB-folds and two Ec-SSB subunits containing one OB-fold per subunit. The linker observed between the two OB-folds in the Dr-SSB protein is shown in red and is the linker used to design the SSB-LD-Drl and SSB-LT-Drl proteins.

Figure 2

A) SDS-PAGE analysis of recombinantly purified wt SSB, SSB-LD-Drl and SSB-LT-Drl proteins. 15 μl of 2 μM protein stocks was analyzed on a 12 % SDS-PAGE gel. B) Sedimentation velocity analysis of wt SSB, SSB-LD-Drl and SSB-LT-Drl proteins at 42000 rpm show the presence of a single species in solution for all three proteins. The SSB-LD-Drl (C) and SSB-LT-Drl (D) proteins sediment as tetramers in equilirium centrifugation experiments with molecular weights corresponding to a single tetramer with 4-OB folds (LD-Drl: 65070 Da and LT-Drl: 61626 Da). The experiments were done using three different protein concentrations (as noted) and at four rotor speeds (9500, 11500, 14000 and 17000 rpm). These experiments were performed at 25 °C in buffer containing 30 mM Tris-Cl, pH 8.0, 10 % glycerol, 0.2 M NaCl and 1 mM EDTA.

Figure 3

ssDNA binding properties of linked SSB tetramers. A) Occluded site-size measurements as a function of increasing [NaCl] for the wt SSB and linked SSB proteins on poly(dT) ssDNA show the presence of three distinct DNA binding modes (SSB)₃₅, (SSB)₅₅, and (SSB)₆₅ for all three proteins. (B) Measurement of occluded site size in replication buffer show that all three proteins bind to ssDNA in the (SSB)₆₅ binding mode. C) Quenching of intrinsic SSB Trp fluorescecene upon binding to a (dT)₇₀ oligonucleotide shows that all three proteins bind stoichiometrically. D) Wrapping of ssDNA around wt SSB and linked SSB proteins measured using a oligonucleotide with Cy5.5 and Cy3 fluorophores positioned at the 5′ and 3′ ends respectively, and monitoring enhancement of Cy5.5 fluorescence at 700 nm by exciting the Cy3 probe at 515 nm. (E) Binding of (dT)₃₅ to wt SSB and linked SSB tetramers show binding of two (dT)₃₅ molecules to wt SSB (K₁ > 10¹⁵ M⁻¹ and K₂ = 1.60 ± 0.16 × 10⁷ M⁻¹) whereas both the SSB-LD-Drl and SSB-LT-Drl tetramers bind to one (dT)₃₅ with high affinity (K₁ > 10¹⁵ M⁻¹ for both SSB-LD-Drl and SSB-LT-Drl) whereas the second (dT)₃₅ binding is weaker (K₂ = 1.66 ± 0.71 × 10⁵ M⁻¹ and 2.34 ± 0.29 × 10⁵ M⁻¹ for SSB-LD-Drl and SSB-LT-Drl respectively). These experiments were done at 25 °C in buffer containing 50 mM Hepes pH 7.5, 10 mM Mg(OAc)₂, 100 mM NaCl, 100 mM KC₅H₈NO₄, and 20 % glycerol.

Figure 4

Linked SSB tetramers with only one C-terminal tail show decreased stimulation of DNA replication. (A) In-vitro single-stranded DNA replication assays were carried out in the presence of indicated SSB derivative. (B) In vitro rolling circle DNA replication assays were carried out in the presence of indicated SSB. (C) The products from the rolling circle replication reactions were fractionated on an alkaline agarose gel and the length of Okazaki fragments were determined. (From left to right: 2775, 2260, 2630, 2145, 2615, and 2145 nt.)

Figure 5

SSB-LT-Drl does not support PriA-dependent replication restart pathway. (A) DNA substrate used in unwinding reactions. The fluorescence of TET on the 5′ terminus increases when separated by helicase action from a quencher (BHQ-1) on the lagging strand template. Streptavidin binding to biotinylated thymidine on the 5′-end of the lagging strand template blocks helicase self-loading by threading over a free 5′-end. There is a 10 nt gap between the 3′-OH of the leading strand primer and the duplex region of the fork. (B) SSB forms titrated individually in triplicate in the presence of 150 nM PriA, 50 nM PriB₂, 50 nM DnaT₃, 12 nM DnaB₆, 50 nM DnaC.

Figure 6

In vivo repair capabilities of E. coli strains carrying wt SSB or ssb-LD-Drl genes. Serial dilutions of cells in the absence or presence of 100 mM hydroxyurea (A) or 2 mM nitrogen mustard (B). Cells harboring the ssb-LD-Drl gene recover better compared to the wt cells in the presence of the either DNA damaging agent. Both strains are tolerate lower levels of UV to similar extents (C). However at a higher dose of UV (D), only the cells carrying the ssb-LD-Drl gene is able to grow. E) Western blot detection of RecA levels in the absence or presence of 100 mM Nalidixic acid. Both strains are capable of inducing RecA expression in the presence of DNA damage.

Figure 7

Growth characteristics of E. coli cells carrying either four or two-tailed SSB tetramers. A) Rifampicin resistance assay showing the frequency of mutations in strains carrying the wt SSB or the ssb-LD-Drl genes. Growth analysis of E.coli cells with the wt ssb gene or ssb-LD-Drl gene shows faster recovery of the two-tailed SSB strain when the cultures are started from a overnight passage (B) or from a log phase starter culture (C).