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. 2020 Aug 18;9:e58622. doi: 10.7554/eLife.58622

Figure 4. Several regions of ORC contact the endogenous DNA in ORCDNA.

(a) ORC subunits 1–5 contain regions with positively-charged residues proximal to the DNA. (b) The ORC1 RecA-fold contains several positively-charged residues in close proximity to DNA. (c) The ORC2 WHD, shown as a density map due to the lower resolution of this domain, moves to interact with the DNA in the core of the complex. (c) The β-hairpin loops of ORC4 and ORC5 contain positively-charged residues proximal to the DNA. The DNA depicted consists of non-specific sequence. (d) The ORC4 β-hairpin loop of S. cerevisiae contains an α-helix that inserts into the major groove of DNA making sequence-specific interactions with DNA bases. (e) The β-hairpin loop in ORCDNA does not contain an α-helix. However, ORC4 R390 in humans may interact with the DNA backbone similar to ORC4 R478 in S. cerevisiae. (f) The superimposition of the ScORC and HsORC β-hairpin loops illustrates the significant difference between the regions.

Figure 4.

Figure 4—figure supplement 1. DNA density observed in the ORC core.

Figure 4—figure supplement 1.

Close-up of the DNA in ORCDNA with mesh density map overlaying the structure from the DNA axis (a) and side view (b). The top panel shows the mesh density map overlaying the structure and the bottom panel shows the surface density map with DNA in red and subunits in close proximity highlighted.
Figure 4—figure supplement 2. Endogenous Sf9 DNA co-purifies with ORC1-5.

Figure 4—figure supplement 2.

(a) After strep-tag purification of ORC1-5, the sample was applied to a 1% agarose gel. The ORC sample contained DNA of less than 0.5 kb. (b) Post 2D classification of the ORCDNA particle population. A strong signal is observed in the core of the complex.
Figure 4—figure supplement 3. The DNA in the core of ORCDNA adopts multiple conformations.

Figure 4—figure supplement 3.

Further 3D classification of ORCDNA particles revealed the DNA in multiple conformations in the core.
Figure 4—figure supplement 4. ORC1 sequence alignment with a focus on the region in close proximity to DNA in the ORCDNA structure.

Figure 4—figure supplement 4.

Sequences are ordered by similarity to H. sapiens. Positively-charged residues in close proximity to DNA in the human ORCDNA structure are highlighted in yellow. The sequence alignment was generated using MUSCLE (Edgar, 2004).
Figure 4—figure supplement 5. ORC4 sequence alignment with a focus on the β-hairpin loop region near DNA.

Figure 4—figure supplement 5.

Sequences are ordered by similarity to H. sapiens. The β-hairpin loop region is highlighted in yellow. ORC4 R390 in humans may interact with the DNA backbone similar to ORC4 R478 in S. cerevisiae. These arginines are highlighted in red. The sequence alignment was generated using MUSCLE (Edgar, 2004) and then structurally aligned.