Similarities between the DNA replication initiators of Gram-negative bacteria plasmids (RepA) and eukaryotes (Orc4p)/archaea (Cdc6p)

Abstract

The proteins responsible for the initiation of DNA replication are thought to be essentially unrelated in bacteria and archaea/eukaryotes. Here we show that RepA, the initiator from the Pseudomonas plasmid pPS10, and the C-terminal domain of ScOrc4p, a subunit of Saccharomyces cerevisiae (Sc) origin recognition complex (ORC), share sequence similarities. Based on biochemical and spectroscopic evidence, these similarities include common structural elements, such as a winged-helix domain and a leucine-zipper dimerization motif. We have also found that ScOrc4p, as previously described for RepA-type initiators, interacts with chaperones of the Hsp70 family both in vitro and in vivo, most probably to regulate the assembly of active ORC. In evolutionary terms, our results are compatible with the recruitment of the same protein module for initiation of DNA replication by the ancestors of present-day Gram-negative bacteria plasmids, archaea, and eukaryotes.

Figure 1

(a) Sequence alignment between pPS10 RepA, ScOrc4p, and PaCdc6p shows similarities between these DNA replication initiators. Identical residues (*) and conservative changes (+) in RepA vs. ScOrc4p alignment are shown. Residues in PaCdc6p found to be identical (24%) in RepA and/or ScOrc4p (black squares). Secondary structural elements are labeled according to the crystal structures of a Rep-type monomer (24) and PaCdc6p (28): the two WH domains in RepA are colored red (WH1) and blue (WH2), whereas that in PaCdc6p is in green. The conserved hydrophobic heptads in the two N-terminal α-helices and Trp-94 (RepA)/Trp-451 (ScOrc4p) are boxed in orange. The alignment between RepA and ScOrc4p was generated with clustalw (29), with minor manual adjustments. After the yeast sequence was shuffled 1,000 times (http://www.ch.embnet.org/software/PRSS_form.html), the probability of a better alignment (score ≥ 294) between RepA and a sequence of the randomized population was 0.0108. Once the sequence of PaCdc6p was available (28), it was included using the alignment as a profile (29). European Molecular Biology Laboratory database accession numbers: RepA, X58896; ScOrc4p, SC34862. (b) Least-squares superposition of the peptide backbones of the WH domains from the RepA homologue RepE54 (24) (Protein Data Bank entry 1REP) and PaCdc6p (28) (1FNN). Forty-one Cα atoms from both three-helix bundle cores (α2-α4 and α16-α18, respectively) were fit with a rms deviation of 2.14 Å. Coordinate transformation was performed with o (30) and displayed with molscript (31).

Figure 2

Structural similarities between RepA and ScOrc4p. (a) Scheme of cloned ScOrc4p (full length and fragments) and SDS/PAGE of the purified proteins (4 μg per lane). (b) FPLC gel filtration profiles for proteins shown in a. Molecular weights and association states assigned to the peaks are indicated: m, monomer; d, dimer; t, tetramer; o, oligomer. Standards (kDa): alcohol dehydrogenase (ADH), 150; BSA, 66; ovalbumin, 45; carbonic anhydrase, 29; cytochrome c, 12. (c) CD spectra of RepA (16) and ScOrc4p-ΔN366. (d) CD thermal denaturation profiles for ScOrc4 proteins: WT, 10 μM; ΔC367, 14 μM; ΔN366, 30 μM; ΔN402, 22 μM. Calculated Tm are indicated. To be compared with the others, the ΔN402 curve is down-shifted by 4.2 × 10³ θ_MR units.

Figure 3

E. coli DnaK binds to ScOrc4p and disassembles oligomers. (a) SDS/PAGE of soluble lysates and Ni²⁺ affinity chromatography peak fractions for proteins in Fig. 2a and RepA. The arrow points to the 70-kDa copurifying band. (Top) Western blot with anti-DnaK polyclonal antiserum. (b) (Top) FPLC gel filtration analysis of purified DnaK-ScOrc4p (ΔC367) complexes (lane L) after incubation with ATP, at 4°C or 37°C. Elution positions of standards (Fig. 2b) and unbound ATP/ADP are marked on the absorption profiles. (Bottom) SDS/PAGE and densitometric ratios for significant fractions.

Figure 4

Yeast Hsp70 binds to ScOrc4p and RepA in vitro. (a) SDS/PAGE of proteins from yeast WCE bound to Ni²⁺-Sepharose saturated with His6-tagged ScOrc4p or RepA (major bands). (Top) Western blot with anti-Hsp70 mAb. (b) Immunoprecipitation with anti-Hsp70 of mixtures of WCE and proteins shown in a. (Center) SDS/PAGE. Unfilled arrows point to IgG heavy (H) and light (L) chains; full-length ScOrc4p, ΔN366, and RepA are hidden under them (see next). (Bottom) Western blot with horseradish peroxidase–anti-His mAb. (Top) Membrane was reprobed with anti-Hsp70 mAb.

Figure 5

ScOrc4p and RepA interact with yeast Hsp70 in vivo. (a) Constructs for the expression of ScOrc4p and RepA in S. cerevisiae. (b) Western blot with anti-HA mAb of chromatin fractions from induced yeasts carrying plasmids shown in a. The asterisk marks a nonspecific band that provides a control for loading. Arrows point to ScOrc4p/RepA products. (c) SDS/PAGE of proteins immunoprecipitated by anti-Hsp70 in WCE from yeast found in b to have ScOrc4 proteins (or RepA) associated with chromatin. (Bottom) Western blot with horseradish peroxidase–anti-His mAb. (Top) The membrane reprobed with anti-Hsp70 mAb.