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. 2013 Feb 21;3(2):485-96.
doi: 10.1016/j.celrep.2013.01.002. Epub 2013 Jan 31.

Specificity and Function of Archaeal DNA Replication Initiator Proteins

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Specificity and Function of Archaeal DNA Replication Initiator Proteins

Rachel Y Samson et al. Cell Rep. .
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Abstract

Chromosomes with multiple DNA replication origins are a hallmark of Eukaryotes and some Archaea. All eukaryal nuclear replication origins are defined by the origin recognition complex (ORC) that recruits the replicative helicase MCM(2-7) via Cdc6 and Cdt1. We find that the three origins in the single chromosome of the archaeon Sulfolobus islandicus are specified by distinct initiation factors. While two origins are dependent on archaeal homologs of eukaryal Orc1 and Cdc6, the third origin is instead reliant on an archaeal Cdt1 homolog. We exploit the nonessential nature of the orc1-1 gene to investigate the role of ATP binding and hydrolysis in initiator function in vivo and in vitro. We find that the ATP-bound form of Orc1-1 is proficient for replication and implicates hydrolysis of ATP in downregulation of origin activity. Finally, we reveal that ATP and DNA binding by Orc1-1 remodels the protein's structure rather than that of the DNA template.

Figures

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Figure 1
Figure 1
Individual Orc1/Cdc6 Genes Are Nonessential (A) Western analyses of Orc1/Cdc6 levels in wild-type (wt) and strains deleted for orc1-1 (Δ1), orc1-2 (Δ2), or orc1-3 (Δ3). Antiserum against PCNA3 was used as a loading control. (B) Growth curves of the indicated strains, following growth by absorbance at 600 nm. (C) Flow-cytometry profiles of WT and mutant strains. Positions of single- and double-chromosome (1C and 2C) content cells are indicated. (D) 2D gel analysis of origin activity in wild-type (wt) and the indicated mutant strains. The position of the bubble arc, indicative of replication initiation, is indicated by a red arrowhead. See also Figures S5, S7, and Tables S1, S2, S3, and S4.
Figure 2
Figure 2
Effect of Deleting Combinations of Orc1/Cdc6 Genes (A) Western analyses of Orc1/Cdc6 levels in wild-type (wt) and strains deleted for orc1-2 and orc1-3 (Δ2+3), orc1-1 and orc1-3 (Δ1+3), or orc1-1 and orc1-2 (Δ1+2). Antiserum against PCNA3 was used as a loading control. (B) Growth curves of the indicated strains, following growth by absorbance at 600 nm. (C) Flow-cytometry profiles of WT and mutant strains. Positions of single- and double-chromosome (1C and 2C) content cells are indicated. (D) 2D gel analysis of origin activity in WT (wt) and the indicated mutant strains. The position of the “bubble arc,” indicative of replication initiation, is indicated by a red arrowhead. (E and F) Marker frequency analysis of wild-type cells (E) and cells lacking Orc1-1 and Orc1-3 (F). Marker ratios of sequence tag abundance across the chromosome of exponentially growing cells normalized to nonreplicating stationary phase cells are plotted relative to genome position. The locations of the three origins are indicated above the plots. See also Figure S7, and Tables S1, S2, S3, and S4.
Figure 3
Figure 3
WhiP Mediates Replication Initiation at oriC3 (A) Western analyses of WhiP in the wild-type (wt) strain and a strain containing a version of whiP with tandem stop codons following the start codon (whip). (B) Growth curves of the indicated strains, following growth by absorbance at 600 nm. (C) Flow-cytometry profiles of WT and mutant strains. Positions of single- and double-chromosome (1C and 2C) content cells are indicated. (D) 2D neutral-neutral agarose gels showing oriC1oriC2, and oriC3 activity in the whip mutant strain. The position of the bubble arc, indicative of replication initiation, is indicated by a red arrowhead. (E) MFA profile of the whip mutant. The positions of the three origins are shown. See also Figures S6, S7, and Tables S1, S2, S3, and S4.
Figure 4
Figure 4
Origin Binding In Vivo by the Orc1/Cdc6 Proteins and WhIP (A–D) ChIP analyses to assess the binding of the indicated initiator proteins to the three origin loci in vivo. The percent of input DNA recovered was calculated for each origin locus and a distal control locus (ctrl, SiRe2328). Bars represent the SD calculated from the triplicate experiments. (E–G) ChIP-seq results with Orc1-1, Orc1-3, and WhiP antisera, respectively. Genome coordinates are shown below each panel and the location of each origin indicated in bold. See also Figure S1. The y axis scale is the value obtained when normalizing ChIP read counts to input DNA. See also Figure S2, and Tables S1, S2, S3, and S5.
Figure 5
Figure 5
Constitutive Expression of Orc1-1 Causes an Overreplication Phenotype (A) Western analysis of Orc1-1 levels in WT cells (lane 1), Δorc1-1 cells (lane 2), the Δorc1-1 strain complemented with a plasmid encoding the gene for Orc1-1 under the control of its native promoter (lane 3), or under the control of the arabinose promoter under noninducing and inducing conditions (lanes 4 and 5). PCNA2 serves as a loading control. Note that in the lower panels the samples in lanes 4 and 5 contain 1/50th the amount of material present in lanes 1–3. (B) 2D gel analyses of oriC1 in the indicated strains. The red arrowheads indicate structures arising from replication initiation. (C) Flow-cytometry analyses of the DNA content of the indicated strains. Peaks corresponding to 1C and 2C cells are indicated. (D) Micrographs of the indicated strains. Phase contrast, cells stained with the membrane dye FM4-64X (red), and nucleoids stained with DAPI (blue) are shown. Yellow asterisks indicate cells extruding vesicle-like structures. (E) Transcript abundance profiles of orc1-1, orc1-2, orc1-3, and whiP as a function of position in the cell cycle. Cells were synchronized using a baby machine and RNA purified as described (Samson et al., 2011). Bars are ±SD. See also Figures S3, S4, S7, and Tables S1, S2, and S4.
Figure 6
Figure 6
Expression of a Walker B Mutant Orc1-1 Causes an Overreplication Phenotype (A) Western analysis of Orc1-1 levels in WT (lane 1) and Δorc1-1 (lane 2) strains and the latter strain complemented with a plasmid encoding the gene for WT Orc1-1 (lane 3), K69A Walker A mutant (lane 4), and E147A Walker B mutant (lane 5) under the control of the native orc1-1 promoter. (B) ChIP analysis of the indicated Orc1-1 protein binding to oriC1 and a distal control locus (Sire_2328) in vivo. Error bars are ±SD. (C) 2D gel analyses of replication initiation at oriC1 in the indicated strains. Red arrowheads indicate structures arising due to replication initiation. (D) Flow-cytometry analyses of the cell-cycle profile of the indicated strains. Positions of 1C and 2C populations are indicated. (E) Micrographs of the indicated strains (+wt = Δorc1-1 + pOrc1-1, +K69A = Δorc1-1 + pOrc1-1 [K69A]; +E147A = Δorc1-1 + pOrc1-1 [E147A]). Phase contrast, membrane dye FM4-64X (red), and DAPI-stained (blue) images are shown. (F) Quantitation of aberrant cells (scored as having a diameter greater than 1.5 μm). Note that large cells in the Walker A strain appear to have less DNA than the large cells in the Walker B strain. See also Figures S3, S4, S7, and Tables S1, S2, and S4.
Figure 7
Figure 7
Biochemical Consequences of the Walker B E147A Mutation (A) Nucleotide-dependent conformational changes in Orc1-1. Gel filtration analysis of 10 μg of WT Orc1-1⋅ADP or the E147A⋅ATP on a Superdex 75 column. The elution positions of size standards are indicated (conalbumin, 75 kDa; ovalbumin, 44 kDa; and ribonuclease A, 13.7 kDa). (B) Composite three-dimensional contour plot of hydrodynamic parameters obtained from GA-MC50 analysis of WT and Walker B mutant Orc1-1 (E147A). WT analysis reveals the presence of two conformers of the monomer, while the Walker B mutant results indicate the presence of a single monomer conformer in addition to a dimer form (see also Figure S3). (C) The structure of the Aeropyrum pernix Orc1-1 ortholog bound to an ORB element from oriC1 is shown. The ISM is shown in red and the wH domain in blue. (Figure generated using Pymol from Protein Data Bank file 2V1U.) DNA contact points are correspondingly color-coded. The structure is aligned to the sequence of the S. islandicus oriC1 ORB2 element. The left-hand gel image shows Fe⋅MPE cleavage patterns in the absence (−) or presence of 1 μM WT or Walker B E147A mutant protein (WB) in the presence of 2 mM ATP (WB). Positions of protection from cleavage are identified at the right of the panel. The right-hand panel shows DNaseI footprinting performed in the absence (−) or presence of 1 μM WT or Walker B E147A mutant protein (WB) in the presence of 2 mM ATP (WB). (D) EMSA analysis of WT or Walker B E147A mutant protein binding to a 231 nt probe containing S. islandicus oriC1. Lanes contain 0, 63, 125, 250, 500, or 1000 nM of the indicated Orc1-1 protein. (E) Trypsin cleavage products following 22 hr digestion of WT (lanes 2, 4, 6, and 8) and Walker B mutant (lanes 3, 5, 7, and 9) in the absence (lanes 2–5) or presence (lanes 6–9) of ORB2 DNA. (F) Identity of the terminal trypsin digestion site by determined by liquid-chromatography tandem mass spectrometry. (G) Western analysis of MCM recruitment to paramagnetic beads containing either control DNA (c) or oriC1-containing DNA (o). Reactions contained DNA at 200 pM and were preincubated with buffer (lanes 3, 4, 9, and 10) or 1 μM WT recombinant protein (lanes 5, 6, 11, and 12) or Walker B mutant E147A Orc1-1 (lanes 7, 8, 13, and 14) in the presence (lanes 9–14) or absence (lanes 3–8) of 2 mM ATP. Following preincubation, extract was added, incubated, and then washed prior to elution of bound proteins by boiling with SDS PAGE loading buffer. 5 and 0.5% input extract were loaded in lanes 1 and 2. MCM was detected by western blotting. See also Figure S4.
Figure S1
Figure S1
Binding of the Three Orc1/Cdc6 Proteins and WhiP on the Three Origins and the orc1-2 Promoter Region, Related to Figure 4 (A–D) ChIP Seq profiles of the 4 proteins binding to the indicated loci. (E) DNaseI footprinting of WhIP binding to sequence repeats within its own ORF. The right panel shows the sequence of the WhiP ORF with a perfect direct repeat indicated in red, runs of four or more A or T residues are indicated by underlining. The gray box indicates the extent of the footprint seen in the left-hand panel.
Figure S2
Figure S2
Analysis of Species Specificity of oriC2 Binding by S. islandicus (Sis) and S. solfataricus (Sso) Orc1-1 and Orc1-3, Related to Figure 4 Both origin sequence and initiator protein sequence define the differential binding of Orc1-1 to oriC2 between S. solfataricus and S. islandicus. (A) Sequence alignment of the oriC2 loci from the two species. Asterisks indicate sequence identity. Mapped binding sites for Sso Orc1-1 (mORB elements, yellow) and Orc1-3 (C3b, blue) are highlighted. As can be seen mORBa in S. islandicus has four points of sequence divergence from Sso including in the region of dyad symmetry (shown by converging arrows). The underlined regions correspond to the oligonucleotides used in the EMSAs in Figures S2B–S2D. (B) EMSAs with 0, 0.5, 1 or 2 μM Sso or Sis Orc1-1 on oligonucleotides corresponding to the mORB regions of S. islandicus oriC2. While The Sso protein binds readily to these sequences, the cognate Sis protein binds with at least 10-fold lower affinity. (C) EMSAs with 0, 0.5, 1 or 2 μM Sso or Sis Orc1-1 on oligonucleotides corresponding to the mORB regions of S. solfataricus oriC2. Sso Orc1-1 binds to the Sso oriC2-derived sequences. Sis Orc1-1 binds with higher affinity to the Sso mORBa sequences than it does to the homologous region of its own species’ oriC2. (D) 0, 64, 128, 256, 512 nM Sis Orc1-3 binds to C3b-containing sequences derived from either Sso or Sis oriC2.
Figure S3
Figure S3
Biochemical Consequences of Walker A and Walker B Mutations of Orc1-1, Related to Figures 5, 6, and 7 (A) 10 μg of refolded WT, Walker A K69A mutant (WA) or Walker (WB) were incubated with 0.375 MBq of α-32P ATP binding. Reactions were split in two and half analyzed by native gel electrophoresis in Tris-glycine, before phosphorimaging of the wet gel (upper panel); the other half was subjected to SDS PAGE (lower panel). (B) ATPase assays were performed with refolded WT or Walker B (WB) E147A mutant protein. Assays contained no DNA, 20 ng of poly(dGdC) or 20 ng of ORB2 double stranded oligonucleotides. (C) AUC profiles of WT and Walker B E147A proteins. Upper panels: Sedimentation velocity data (dotted line) and model fit results (solid line) for WT and Walker B Orc1-1. Lower panels: Residuals obtained from 50 iterations of Genetic Algorithm-Monte Carlo analysis of the data set, based on the solution of the hydrodynamic Lamm equation.
Figure S4
Figure S4
Model for Consequences of ATP Binding by Orc1-1, Related to Figures 5, 6, and 7 (A) Structure of the ADP-bound form of Orc1-1 from Aeropyrum pernix bound to DNA (from PDB code 2V1U). The wH domain is shown in blue, the ISM within the AAA+ domain in red and the ADP molecule in yellow. The protein contacts DNA via wH and ISM. (B) A model of the ADPNP–bound form of the Aeropyrum Orc1-2 homolog (Singleton et al., 2004)on DNA. This model was generated by aligning the Orc1-2 protein to the DNA-bound form of Orc1-1 via the wH domain. Color coding is as in part A with ADPNP shown in yellow. (C) Potential scenarios for the effect of ATP binding by Orc1/Cdc6 protein suggested by the model above. Model 1. ATP binding causes the ISM to disengage from the DNA. An extension of this model is shown in Model 1a where the protein is now able to multimerise into a filament-like structure via the AAA+ domain. This scenario is related to the behavior of DnaA (Erzberger et al., 2006, Duderstadt et al., 2011). In Model 2 both ISM and wH remain bound to DNA in the ATP-bound form, resulting in an alternate conformation of the DNA to that seen in the presence of ADP. Finally, in Model 3 both ISM and wH remain engaged with DNA but the DNA conformation is unaltered but the protein remodels itself relative to the ADP-bound state.
Figure S5
Figure S5
Plasmid Maps for Constructs Used to Generate Knockouts, Related to Figure 1
Figure S6
Figure S6
Schematic of the strategy employed to generate the Whip mutant cell line, Related to Figure 3
Figure S7
Figure S7
2D Gel Strategy, Related to Figures 1, 2, 3, 5, and 6 Diagram of the loci encoding (A) oriC1, (B) oriC2 and (C) oriC3. Restriction sites and positions of the sequences utilized to generate the radiolabelled probes employed in the 2D gel analyses are indicated. The position of the replication origin in each region is indicated by a circle.

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