Fig 1.
A model of bacterial linear DNA replication.
Replication of the linear DNA molecule initiates at an internal origin and proceeds bidirectionally through the hairpin telomeres. This produces a circular dimer of replicated DNA fused at replicated telomere junctions containing inverted repeat symmetry (denoted at L/L’ and R/R’). The replication intermediate is then resolved into two linear replicons closed by hairpin ends through a process called telomere resolution.
Fig 2.
A) A schematic representation of the singled-stranded annealing assay with naked DNA. The diagram shows the two complementary 87-nt oligonucleotides (OGCB455/456), with the 5’-32P endlabeled oligonucleotide represented in red and the unlabeled oligonucleotide shown in black. The 5’-32P endlabel is shown with the red asterisk. Addition of TelA to the reaction (blue squares) stimulates annealing of the two complementary oligonucleotides. B) Representative gel panels of 8%PAGE/1X TAE/0.1% SDS gel analysis for timecourse annealing reactions including spontaneous annealing (no protein), wildtype TelA, and the telomere resolution active site nucleophile mutant of TelA, TelA (Y405F). Where protein is present, gel panels using 135 nM of TelA are shown. The migration positions of the 5’-32P endlabeled reporter oligonucleotide (ss) and the duplex product (ds) are labeled. The electrophoretic mobility of native PAGE is affected by size, charge, and shape. The slower mobility of ss in this figure as compared to ds is likely due to the presence of secondary structure making ss less compact. C) A plot of annealing rate vs. TelA concentration is shown comparing wildtype TelA and the telomere resolution active site nucleophile mutant. The mean and standard deviation are shown and are derived from three independent experiments. Where error bars are not apparent the standard deviation was smaller than the points plotted.
Fig 3.
Determination of ssDNA length requirements for TelA-promoted single-stranded annealing.
A) A schematic representation of the 69 nt 5’-32P endlabeled reporter oligonucleotide (red) and a set of eight unlabelled partner oligonucleotides (black) used in the single-stranded annealing assay. Oligonucleotide 1 is a fully complementary partner to the reporter oligo that anneals to form a 65-nt duplex with GATC 5’-overhangs. Each successive oligonucleotide introduces an additional 6-nt block of non-complementary sequence (highlighted in green), asymmetrically positioned to produce annealed products with different arm lengths and varying melting points. These are displayed to the right of the schematic. B) 8% PAGE/ 1X TAE/ 0.1% SDS gel panels of the spontaneous annealing (w/o TelA) and TelA-promoted annealing assays. The single-stranded annealing assays were performed as described in the Materials and Methods with each reaction being incubated for 20 s at 30°C prior to reaction termination and gel loading. S represents the migration position of the single-stranded reporter oligonucleotide. Bubble products are duplex products that are annealed at both ends despite lacking central base pairing, and frayed-end products are anchored at their left end only. The gel migration breakpoint between bubble products and frayed-end products was determined by observing the migration position of the reaction products in comparison to a modified version of oligo 5 that possesses only left flank homology and 41-nt of non-complementary sequence and thus, can only anneal into a fray-end product (see OGCB478 in S1 Table).
Fig 4.
TelA anneals ssDNA possessing secondary structure.
A) A schematic representation of the single-stranded annealing assay using substrates with complex secondary structure. These complementary DNA oligonucleotide sequences (red and black) are a mimic of an HIV transactivational response (TAR) element and naturally form a complex stem-loop structure with several bulges in their single-stranded form. Addition of TelA (blue squares) promotes removal of the secondary structure and subsequently stimulates annealing of the complementary strands into a lineform duplex. B) Representative 8% PAGE/ 1X TAE/ 0.1% SDS gel panels of timecourse annealing reactions with TAR sequences with or without TelA present. Where protein is present, reactions using 154 nM of TelA are shown. The migration positions of the 5’-32P endlabeled reporter TAR oligonucleotide (ss) and the duplex product (ds) are labeled. The electrophoretic mobility of native PAGE is affected by size, charge, and shape. Here ss migrates faster than ds, a contrast to Fig 2B. The TAR ss possesses significant internal base pairing making it extremely compact. C) A plot of annealing timecourses comparing spontaneous annealing and TelA-promoted annealing with TAR sequences. The mean and standard deviation are shown and are derived from three independent experiments.
Fig 5.
TelA can anneal plasmid length ssDNA.
0.7% agarose/ 1X TAE gel panels showing plasmid annealing reactions. The positions of heat denatured pUC19 (ssDNA), unit-length plasmid duplex (dsDNA) and the wells are labeled to the right of the gel. Reaction timepoints and the presence or absence of reaction components are indicated in the key above the gel. 76 nM of TelA was incubated with 1.78 mM nucleotides of the plasmid substrate in buffer containing 25 mM HEPES (pH 7.6), 2 mM MgCl2, 1 mM DTT, 100 μg/mL BSA and 50 mM potassium glutamate. pUC19 was linearized, 32P-endlabeled and denatured as described in the Materials and Methods section.
Fig 6.
TelA promotes limited DNA strand exchange.
A) A schematic representation of the DNA strand exchange and control reactions with a 5’-32P endlabeled 43-bp partially duplexed target (410*/409) and a 63-nt ssDNA homologous donor (411, blue) with 20 nt of 3’ flanking homology with the bottom strand of the partial duplex target or 63 nt of ssDNA of randomized sequence (426, green). The top strand of the partial duplex is red and the red asterisk represents the 5’ end label; the bottom strand is represented in black. B) 8% PAGE/ 1X TAE/ 0.1% SDS gel analysis of timecourse strand exchange reactions with combinations of TelA and various donors as indicated above the gels. The migration position of the partial duplex is labelled as 410*/409 and the migration of the displaced strand if strand exchange occurs is labelled as 410*.
Fig 7.
TelA promotes annealing with ssDNA complexed with its cognate single-stranded binding protein.
A) A schematic representation of annealing assays with naked DNA and SSB-complexed DNA. Details of the experimental set up are as previously described in the legend to Fig 2 but with the addition of annealing reactions with SSB-complexed ssDNA. For annealing reactions with SSB-complexed DNA, the two complementary oligonucleotides are separately preincubated with SSB (green ovals). They are then mixed, followed by addition of TelA to initiate the annealing reaction. B) Plots of annealing timecourses comparing naked DNA and SSB-complexed DNA, with or without added TelA. Two plots are shown to represent timecourses performed with TelA concentrations below (38 nM) and above (115 nM) the equivalent molar concentration of added SSB (105 nM). The mean and standard deviation are shown and are derived from three independent experiments. C) Plots of annealing timecourses comparing TelA with SSB-complexed DNA (blue) and SSBΔC7 complexed DNA (red), with or without TelA. Two plots are shown to represent timecourses performed with TelA concentrations below (76 nM) and above (154 nM) the equivalent molar concentration of added SSB (105 nM). The mean and standard deviation are shown and are derived from three independent experiments.
Fig 8.
TelA promotes annealing of plasmid length DNA complexed with its cognate SSB.
0.7% agarose/ 1X TAE gel panels showing SSB-complexed plasmid annealing reactions. The migration patterns of heat denatured pUC19 (ss) and the unit-length plasmid duplex (ds) are labeled as shown. Reaction timepoints and the presence or absence of reaction components are indicated in the key above the gel. 1.78 mM nucleotides of heat denatured pUC19 was preincubated with 105 nM of SSB in buffer containing 25 mM HEPES (pH 7.6), 1 mM DTT, 2 mM CaCl2, and 50 mM potassium glutamate. Following pre-incubation with SSB, annealing was initiated by the addition of 154 nM of TelA.
Fig 9.
Deletion of the N-terminal domain of TelA abolishes TelA’s annealing activity.
A) Representative 8% PAGE/ 1X TAE/ 0.1% SDS gel panels of annealing assay timecourses comparing reactions of naked ssDNA with and without 154 nM TelA (107–442), and SSB-complexed ssDNA with and without 154 nM TelA (107–442). The migration positions of the 5’-32P endlabeled reporter oligonucleotide (ss) and the duplexed product (ds) are indicated. B) Plots of annealing timecourses with naked DNA, SSB-complexed DNA, or SSBΔC7 complexed DNA as labeled. Each plot compares the spontaneous rate of annealing, TelA (107–442) promoted annealing, and wildtype TelA promoted annealing under the indicated conditions. All reactions shown were performed with 154 nM of TelA, and 105 nM of SSB or SSBΔC7 where appropriate. C) A plot of annealing rate vs. TelA (107–442) concentration is shown comparing annealing with naked DNA, SSB-complexed DNA, and SSBΔC7 complexed DNA. The mean and standard deviation are shown and are derived from three independent experiments.
Fig 10.
TelA (1–106) is an annealing protein.
A) Representative 8%PAGE/ 1X TAE/ 0.1% SDS gel panels of annealing timecourses comparing spontaneous annealing (no protein) to reactions containing 288 nM of the N-terminal domain of TelA (TelA (1–106)). The migration positions of the 5’-32P endlabeled reporter oligonucleotide (ss) and the duplex product (ds) are indicated. B) A plot of annealing rate vs. TelA concentration is shown comparing the annealing rates of wildtype TelA (blue) and TelA (1–106) (red). The wildtype TelA data ranging from the spontaneous rate of annealing to 154 nM of TelA was previously shown in Fig 2C. Data for annealing rates with higher concentrations of wt TelA (192 nM– 385 nM) were collected in tandem with TelA (1–106) annealing rates and have not been previously shown. The mean and standard deviation are shown and are derived from three independent experiments.