Figure 5. Dissection of the role of the NTD of Pch2 in origin recognition complex (ORC) association.

(A) Yeast two-hybrid analysis between Orc1 and NH₂-terminal fragments of Pch2 (2-270, 2-257, 2-242, 2-233, 2-194, 2-144, 2-121, 2-91, 2-60, 2-27). Red-dotted box indicates the minimal fragment of Pch2 that showed interaction with Orc1. (B) Size exclusion chromatography of His–MBP–Pch2-2-144 purified from insect cells; Coomassie Brilliant Blue (CBB) staining of the peak fractions (dotted line). AU, arbitrary units. (C) Amylose-based pulldown of the ORC (His–Orc1-6) purified from insect cells, with His–MBP–Pch2 or His–MBP–Pch2-2-144; Coomassie Brilliant Blue staining. (D) Amylose-based pulldown of the ORC (His–Orc1-6) purified from insect cells, with His–MBP–Pch2 or His–MBP–Pch2-2-144; Western blot analysis using α-MBP and α-ORC. (E) Schematic of interaction mode between the ORC and Pch2. Red-dotted box indicates the NH₂-terminal 2-144 region of Pch2’s NTD. Cross-linked residues within the Pch2-2-144 region are indicated with *.