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Science 31 August 2007:
Vol. 317. no. 5842, pp. 1213 - 1216
DOI: 10.1126/science.1143664
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Reports

Structural Basis of DNA Replication Origin Recognition by an ORC Protein

Martin Gaudier, Barbara S. Schuwirth, Sarah L. Westcott and Dale B. Wigley*

Cancer Research UK Clare Hall Laboratories, London Research Institute, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3LD, UK.


Figure 1 Fig. 1. (A) Organization of the Ori1 replication origin in A. pernix. The four ORB sequences are located on either side of the DUE. The sequence of the 5'-to-3' ("top") strand of ORB4 is shown. (B) Overall structure of the ORC1-DNA complex. (C) Contacts between the AAA+ domain and DNA. Thr122 (T122) (21) contacts the Gd18-Cd5 base pair, L124 main chain oxygen contacts Gd19, and E128 contacts Gd20 via a water molecule. Residues T103, R106, G123, and R132 make direct interactions with the phosphodiester backbone on either side of the minor groove. (D) Insertion of the wing of the WH domain into the DNA minor groove widens it by 5 Å. Residues S366, G368, G371, and K372 interact directly with bases Td17, Gd18, and Gd19 of the complementary strand. G368, G371, K372, and T373 also interact with the DNA backbone on both sides of the minor groove. (E) Insertion of the recognition helix of the WH domain into the major groove. R345 makes a base-specific interaction with Gd10. Residues T343, R346, S348, and R374 contact the DNA phosphate backbone. The R346 side chain is stabilized in a noncanonical conformation by a salt bridge interaction with E353, which enables it to bind the DNA phosphate backbone. In the same way, interaction between S352 and R374 brings the arginine side chain close to the DNA backbone. [View Larger Version of this Image (62K GIF file)]
 

Figure 2 Fig. 2. Deformation of the ORB element compared with regular B-form DNA. (A) A comparison between B-form DNA (top) and the ORB4 DNA in the structure (below). The helical axis of the DNA is shown as a blue (B-form DNA) or red (ORB4 DNA) bar running down the center of the duplex. Overall, the DNA is underwound as shown by the twist, which averages 33° per base pair, giving 11 bp per helical turn. DNA parameters were calculated with the program 3DNA (22). (B) Comparison of the base pair roll for each base pair. The roll is greater for all but one base pair in the ORB4 DNA. (C) Comparison of the major and minor groove widths of the ORB element with those for B-form DNA. The minor groove is wider at every position but one along the ORB4 DNA, and the major groove is also wider in all but two places. [View Larger Version of this Image (29K GIF file)]
 

Figure 3 Fig. 3. The stoichiometry of ORC1 binding at the ORB4 element. (A) Isothermal calorimetry data collected by using purified WH domain protein and a 40-mer DNA containing an ORB4 element. (B) Figure mapping the footprint data [(C)] onto the crystal structure. (C) Deoxyribonuclease I footprints across the ORB4 region. DNA sequences are indicated at the side for reference, with the ORB4 sequence boxed. [View Larger Version of this Image (63K GIF file)]
 

Figure 4 Fig. 4. ORC1 proteins bound to ORB elements flanking a DUE would be oriented to place the WH domains, which interact with the MCM helicase, facing the DUE and hence the replication start site. [View Larger Version of this Image (24K GIF file)]
 

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