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. 2004 Sep 9;32(16):4821-32.
doi: 10.1093/nar/gkh819. Print 2004.

Biochemical Characterization of Cdc6/Orc1 Binding to the Replication Origin of the Euryarchaeon Methanothermobacter Thermoautotrophicus

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Biochemical Characterization of Cdc6/Orc1 Binding to the Replication Origin of the Euryarchaeon Methanothermobacter Thermoautotrophicus

Stephanie A Capaldi et al. Nucleic Acids Res. .
Free PMC article

Abstract

Archaeal cell division cycle protein 6 (Cdc6)/Origin Replication Complex subunit 1 (Orc1) proteins share sequence homology with eukaryotic DNA replication initiation factors but are also structurally similar to the bacterial initiator DnaA. To better understand whether Cdc6/Orc1 functions in an eukaryotic or bacterial-like manner, we have characterized the interaction of two Cdc6/Orc1 paralogs (mthCdc6-1 and mthCdc6-2) with the replication origin from Methanothermobacter thermoautotrophicus. We show that while both proteins display a low affinity for a small dsDNA of random sequence, mthCdc6-1 binds tightly to a short duplex containing a single copy of a 13 bp sequence that is repeated throughout the origin. Surprisingly, sequence comparisons show that this 13 bp sequence is a minimized version of the Origin Recognition Box element found in many euryarchaeotal origins. Analysis of mthCdc6-1 mutants demonstrates that the helix-turn-helix motif in the winged-helix domain mediates the interaction with this sequence. Association of both mthCdc6/Orc1 paralogs with the duplex containing the minimized Origin Recognition Box fits to an independent binding sites model, but their interaction with longer DNA ligands is cooperative. Together, our data provide the first detailed biophysical characterization of the association of an archaeal DNA replication initiator with its origin. Our observations also indicate that the origin-binding properties of Cdc6/Orc1 proteins closely resemble those of bacterial DnaA.

Figures

Figure 1
Figure 1
Comparison of M.thermoautotrophicus and E.coli origins of replication. (A) Schematic representation of the origins from M.thermoautotrophicus and E.coli. The M.thermoautotrophicus origin contains a central A/T-rich element flanked by 13 bp repeats spaced at approximately regular intervals along the origin (white arrows). In E.coli, the 260 bp origin contains five high-affinity A/T-rich DnaA boxes (white arrows) adjacent to an A/T-rich region. For both origins, repeats are present on the sense and antisense strand as indicated by the direction of the arrows. (B) The sequence of the repeats in both origins (32,64).
Figure 2
Figure 2
Binding of the mthCdc6/Orc1 homologs to the single and random sites. Binding data for single (open circles) and random (open triangles) site DNA oligonucleotide duplexes are shown for mthCdc6-1 (A) and mthCdc6-2 (B). The open points on the plot are the averaged values from three independent binding experiments with the standard deviation indicated by the error bars. For mthCdc6-1, the solid lines through the data are the best fit to the independent binding sites model (49). Data for mthCdc6-2 binding to the single site also fit to the independent binding sites model, while a smooth line is drawn through the points for the interaction between mthCdc6-2 and the random site.
Figure 3
Figure 3
Position of mutations in the WHD. Ribbon representation of P.aerophilum Cdc6/Orc1 WHD, with the HTH and wing (W) regions labeled and shaded red (39). The position of the M.thermoautotrophicus recognition helix mutations in the WHD based on homology modeling are shown in orange. The location of wing mutations are not shown since this region of the domain is disordered in the P.aerophilum structure (red dots).
Figure 4
Figure 4
SDS–PAGE analysis and CD spectra of mthCdc6/Orc1 variants. (A) SDS polyacrylamide gel (12%) of mthCdc6-1 variants and wild-type mthCd6-1 and mthCd6-2 used in filter binding studies. (B) CD spectra wild-type mthCdc6-1 (gray triangles) compared to R334A/R335A (black circles).
Figure 5
Figure 5
Binding of mthCdc6-1 WHD mutants to the single site. (A) Binding curves for the recognition helix mutants N341A/E342A (black), S330A/S332A (dark blue), R334A/R335A (dark green) and for comparison wild-type protein (red). All curves have been fitted to the independent binding sites model. (B) Binding curves for the wing mutants S355A (light blue), K360A (light green), R362A (purple) and R358A (gray) have also been fitted to the independent binding sites model.
Figure 6
Figure 6
Binding of mthCdc6-1 and mthCdc6-2 to the triple repeat and 282mer. Binding of mthCdc6-1 to the triple repeat (A) and random 282mer (B) fitted to the Hill equation (49). In (A) the inset shows the binding of mthCdc6-1 to the triple repeat on a semi-log plot fitted to the Hill equation (solid line) and independent binding sites model (dashed line). The inset in (B) is an enlarged view of the data for the binding of mthCdc6-1 to the random 282mer at protein concentrations <1000 nM. (C) Binding of mthCdc6-2 to the triple repeat (open circles) and 282mer (open triangles) have also been fitted to the Hill equation.

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