Function of a strand-separation pin element in the PriA DNA replication restart helicase

Abstract

DNA helicases are motor proteins that couple the chemical energy of nucleoside triphosphate hydrolysis to the mechanical functions required for DNA unwinding. Studies of several helicases have identified strand-separating "pin" structures that are positioned to intercept incoming dsDNA and promote strand separation during helicase translocation. However, pin structures vary among helicases and it remains unclear whether they confer a conserved unwinding mechanism. Here, we tested the biochemical and cellular roles of a putative pin element within the Escherichia coli PriA DNA helicase. PriA orchestrates replication restart in bacteria by unwinding the lagging-strand arm of abandoned DNA replication forks and reloading the replicative helicase with the help of protein partners that combine with PriA to form what is referred to as a primosome complex. Using in vitro protein-DNA cross-linking, we localized the putative pin (a β-hairpin within a zinc-binding domain in PriA) near the ssDNA-dsDNA junction of the lagging strand in a PriA-DNA replication fork complex. Removal of residues at the tip of the β-hairpin eliminated PriA DNA unwinding, interaction with the primosome protein PriB, and cellular function. We isolated a spontaneous intragenic suppressor mutant of the priA β-hairpin deletion mutant in which 22 codons around the deletion site were duplicated. This suppressor variant and an Ala-substituted β-hairpin PriA variant displayed wildtype levels of DNA unwinding and PriB binding in vitro These results suggest essential but sequence nonspecific roles for the PriA pin element and coupling of PriA DNA unwinding to its interaction with PriB.

Keywords: DNA helicase; DNA repair; DNA replication; DNA-protein interaction; DNA damage response; protein complex; protein cross-linking; DNA replication restart; DNA strand-separation mechanisms; PriA; primosome assembly; strand-separation pin.

Figure 1.

The PriA CRR contains a β-hairpin at a similar location to the β-hairpin/strand-separation pin elements of RecQ helicases. A, structure (left) and domain architecture (below) of K. pneumoniae PriA (3′-binding domain, winged helix domain, helicase lobe 1, helicase lobe 2, cysteine-rich region, C-terminal domain; PDB 4NL4; (13)) with the DNA from the RecQ/ssDNA–dsDNA co-crystal structures (bacterial RecQ in gray; PDB 4TMU; (32) and RecQ1 in black; PDB 2WWY; (30)) based on structural superimpositions of the helicase cores of the two proteins. Domains of PriA are colored as in domain architecture, with ADP (red sticks) and zinc (gray spheres), and the DNA from the RecQ structures shown (gray and black). 3′-5′ Label above denotes the direction of the ssDNA stretch and PriA helicase directionality. Right two panels: close-up views of the PriA CRR and β-hairpin within the CRR, showing the residues around the loop in sticks and labeling the four residues of the loop (E. coli PriA numbering). DNA (gray as in A; PDB 4TMU) from the bacterial RecQ/ssDNA–dsDNA structure was used in close-up models because this DNA was more continuous than that with RecQ1. B, sequence around the E. coli PriA CRR β-hairpin. Arrows denote β-strands. Dotted lines show Cys (gray)-zinc coordination. The corresponding sequences of the variants used in this study are aligned below. Red letters highlight duplicated/inserted sequence. Blue A depicts Ala substitutions. Yellow X denotes location of codon mutation and Bpa incorporation.

Figure 2.

PriA–DNA fork cross-linking localizes the CRR β-hairpin to the DNA interface with the template-lagging strand. PriA–Bpa variants (3.5 nm) were incubated with a synthetic 4-strand DNA fork (1 nm; depicted in top right) that had each of the four oligonucleotides individually 5′-³²P-labeled and exposed to UV. These samples were SDS denatured and resolved by PAGE (first set of four lanes for each PriA variant: 1) template-lagging strand labeled; 2) template-leading strand labeled; 3) nascent lagging strand labeled; 4) nascent leading strand labeled). These samples were additionally heat-denatured in the presence of trap oligonucleotide (excess unlabeled version of the ³²P-labeled oligonucleotide) for better resolution of the single oligonucleotides (second set of four lanes for each PriA variant). WT PriA is the negative control and all lanes were compared back to these due to the presence of partial reannealing. PriA E492_Bpa is a positive control (20). Arrows highlight the significant shifted bands due to covalent PriA–DNA cross-links. See previous study for expected PriA–DNA cross-link shifts on each strand (20). Representative gel of a single replicate shown out of three.

Figure 3.

PriA–DNA fork cross-linking maps the CRR β-hairpin to the location of DNA strand separation. A, scheme of primer extension analysis of PriA-DNA cross-linked samples from Fig. 2, using a 5′-³²P-labeled primer for the template-lagging strand (red arrow). B, scheme of PriA–DNA fork model (top) and close-up view of PriA-lagging arm model constructed by superimposition of the E. coli PriA structure (PDB 6DGD (14)) helicase core with that of RecQ bound to ssDNA–dsDNA (gray) (PDB 4TMU (32)). Sites of Bpa incorporation are shown in sticks and labeled. Zinc ions are shown as gray spheres. C, urea PAGE of primer extension products from cross-linking samples with the indicated PriA variants (labeled above lane). Gel panel of the region of significant premature primer extension termination bands (highlighted by vertical lines to left), which are labeled to the right with sequence identity corresponding to D. Sequencing ladder (left) was generated using ddC, ddT, ddG, and ddA, respectively, and labeled with the primer extension product that generated that band. Representative gels shown are of at least three replicates. D, sequence around the fork junction of the DNA fork used in cross-linking assays. Dotted lines from the indicated variants mark the significant UV-dependent primer extension blocked bands from C across replicates.

Figure 4.

The CRR β-hairpin is required for the PriA DNA unwinding mechanism. A, PAGE resolved helicase products from incubation of the indicated PriA variants (0.1, 1, and 10 nm) with a synthetic DNA fork (1 nm; shorter version of that used in Figs. 2 and 3; 60-bp duplex and 38-nucleotide arms) composed of four strands (left) or two strands (right). Initial substrates are depicted in black to left of the gel panel and helicase products are depicted in gray. Red star notes location of the 5′-³²P label. The two gel panels are from one experiment of at least three replicates. B, DNA-binding assay following the increase in anisotropy upon increasing levels of PriA binding to a 5′-fluorescein-labeled two-strand DNA fork (1 nm; same fork as in A with green star noting location of 5′-fluorescein). Mean ± S.D. (error bars) of three replicates are fit to a single-site specific model (see “Experimental procedures”). Error bars are obscured by data point when not visible. Parameters of fit ± S.D. are listed in the inset. C, PriA DNA-dependent ATP hydrolysis rates upon incubation with increasing amounts of ssDNA (dT₂₈). Data are mean ± S.D. (error bars) of three replicates. Data were fit (solid line) to obtain parameters ± S.D. listed in the inset.

Figure 5.

The CRR β-hairpin is required for PriA function in vivo. A, microscopy images of some of the strains used in this study (Table S2). Phase-contrast (left column) shows the cell area. GFP fluorescence (second column) was used as a measure of SOS induction, as a fusion reported of the gfp gene to the sulA promoter was used. mCherry fluorescence (right column) was used to visualize the nucleoid through a translational fusion of the hupA gene to mcherry. Scale bar is 10 μm. B, quantification of microscopy images similar to those in A. Relative fluorescence intensity (RFI) of green fluorescent protein (GFP) signal. WT nucleoids are indicated as positive “+” for chromosome partitioning, whereas irregular/elongated nucleoids are indicated as negative “−” for chromosome partitioning. Average cell area (number of pixels) was quantified as a measure for elongated cells. Data are mean ± S.D.

Figure 6.

The CRR β-hairpin serves a role in PriB interaction with the PriA–DNA complex. Native PAGE of EMSA of the synthetic DNA fork (1 nm; depicted to left of gel; used in Fig. 4 except nascent leading strand was included and nascent lagging strand excluded) incubated with PriA variant (2 nm) and increasing concentrations of PriB (0, 10, 40, and 160 nm monomers). First lane is DNA fork alone. Second lane is PriB (160 nm) without PriA. Representative gel of three replicates.

Figure 7.

The CRR β-hairpin serves a physical role in DNA-unwinding without sequence requirement. Quantification of PAGE of helicase products, as described in the legend to Fig. 4A, from incubating PriA (0.075, 0.15, 0.3, 0.6, 1.2, and 2.4 nm PriA) with a 1 nm 4-strand DNA replication fork (top), which PriA variants specifically unwound the nascent lagging strand or two-strand DNA fork (bottom). Inset depicts substrate (black) and product (gray). Fraction unwound was determined by the quantification of the product band over the total quantification of the lane. Data were fit (left panel, solid lines). Right panels quantified PriA (1.2 nm) helicase activity under SSB (250 nm monomers) or PriB (10 nm monomers) stimulation for the 4-strand (top) or 2-strand DNA fork (bottom). Data are mean ± S.D. of three replicates.

Figure 8.

Model of CRR β-hairpin function within the PriA–PriB replication restart pathway. A, PriA domains are colored as described in the legend to Fig. 1A. (1) an abandoned DNA replication fork is created. (2) PriA binds and interacts with the three arms of a DNA replication fork (14) positioning the CRR β-hairpin (black outlined triangle) near the incoming dsDNA. (3) Helicase lobe 1 and 2 domain movements (20, 56) and PriA affinity for fork junctions may result in lobe 2 binding DNA in an extended form and “pulling” the DNA across the CRR β-hairpin. (4) We propose PriB (dimer; magenta) interacts near the CRR β-hairpin/DNA interface. (5) At this location, PriB could bind ssDNA created by PriA helicase activity and stimulate PriA helicase activity through this interaction and/or stabilization of the CRR β-hairpin wedge. (6) PriB hands off its site on PriA and the DNA to DnaT (trimer; cyan) (23, 57, 80, 81). (7) The PriA–DnaT complex hands off the ssDNA site at the fork junction to the replicative helicase DnaB (hexamer; red), with the help of the replicative helicase loader DnaC (yellow) (52, 64). B, (1b) on forks with ssDNA gaps where SSB (tetramer; gray) is bound, (2b) PriA interacts with the SSB tail (light red; binding site on opposite face of PriA, circled by dotted line) and remodels the DNA-binding modes of SSB in a manner that could expose ssDNA (13). (3b) The role of the CRR β-hairpin in unwinding the template lagging arm could additionally function to promote exclusion of proteins from the DNA, such as SSB. (4–7) PriB binding and possible β-hairpin stabilization/helicase stimulation could further promote this process and facilitate ssDNA hand-off and restart pathway progression.