Overall structural features of the gMCM8/9 hexamer.

(A) Domain organization of chicken MCM8 and MCM9. (MCM8, greencyan; MCM9, slate), N-C linker, linkers connecting the NTD and CTD of MCM8 or MCM9; WH, winged helix domain; HTH, helix-turn-helix; the C-tail domain is not included in this study. (B) Fold of the gMCM8/9 dimer. The N-C linkers are shown as dotted lines and the OB hairpins OB-hp are highlighted in orange (MCM8) and yellow (MCM9), while the ACL are highlighted in purple (MCM8) and red (MCM9). The long helix of MCM8 was marked as α5. (C) Ribbon diagram showing the top and side views of gMCM8/9 hexamer with three-fold symmetry axis.

Cryo-EM 3D reconstruction and refinement of the gMCM8/9 complex.

The assembly analysis of the gMCM8/9 NTD ring.

(A) The cutoff structure of the gMCM8/9 NTD presents in cartoon. The dimer interface and trimer interface were indicated by boxes with dotted line and solid line, respectively. (B, C) The dimer interface (B) and trimer interface (C) were mediated by hydrophobic interaction and polar interactions. The interaction details between gMCM8 and gMCM9 in two interfaces are shown in stereo view.

Structural comparison of gMCM8/9 with MCM2-7-containing intermediates.

(A) The gMCM8/9 hexamer (greencyan and slate) was aligned to MCM2-7 hexamer (lightpink) from the OCCM complex. The N-tier rings comparison are presented here with a slightly rotation beginning at MCM5-MCM2 “Gate” and the following MCM6 and MCM4. The “Gate” was indicated by the black arrow. (B, C) Structural superposition of the gMCM8/9 hexamer to the MCM2-7 double hexamer (wheat) by aligning their respective C-tier ring. The bottom view (B) and top view (C) are shown respectively. Note the ∼30° clockwise rotation of the gMCM8/9 N-tier ring comparing to that of MCM2-7.

HROB induces conformational change of hMCM8/9 complex.

(A) Domain organization of human HROB. PRR, proline-rich region, grey; MBM, MCM8/9 binding motif, dark grey; OB, OB fold domain, yellow. The HROB-CTD consisting of the HROB-MBM and OB domain is indicated. (B) The OB domain is required for MCM8/9 helicase in DNA unwinding. Representative autoradiograph of the DNA unwinding reaction conducted using a 32P-labeled ssDNA oligo annealed to the M13mp18 as DNA substrate in the presence of HROB-MBM or HROB-CTD with purified hMCM8/9. Control, DNA substrate without proteins. (C) HROB-CTD interacts with MCM8/9-NTD. Detection by western blot of MCM8/9-FL or MCM8/9-NTD co-precipitated by bead-bound GST or GST-HROB. (D) The diagram of gel filtration shows the co-purified protein complex of HROB-CTD and MCM8/9-NTD. Their complex also indicated in SDS-PAGE. (E) Reconstructed cryo-EM map of MCM8/9 NTD ring in Conformation II. MCM8, greencyan; MCM9, slate. The shape of the channel is oval. (F) The structure of hMCM8/9-NTD from previous study. MCM8/9, orange. (G-J) Structural superposition of the hMCM8/9-NTD in Conformation I and Conformation II. The translations of the Zinc fingers were showed in (H). The ∼5 Å expansion of the trimer interface of hMCM8/9-NTD in Conformation II was shown in (I). The shift distance and angles of MCM8/9 heterodimer were also shown in (I) and (J), respectively.

Cryo-EM 3D reconstruction and refinement of the hMCM8/9 NTD ring.

The spatial distribution of OB fold hairpins and their functional roles in DNA unwinding.

(A, B) Top and side views of the structure of the gMCM8/9 NTD ring. OB-hps of gMCM8 and gMCM9 are highlighted in orange and yellow, respectively (A); ACLs are highlighted in purple and red as indicated (B). (C) Contacts between OB-hps and forked DNA illustrated through fitting a forked DNA fragment into the hexameric gMCM8/9 central channel. While encircling the DNA, the OB-hps of the gMCM9 form the upper layer and that of gMCM8 constitute the lower layer. (D) Sequence alignments of the OB-hps of MCM8 and MCM9 from different species. h, human; m, mouse; g, chicken (Gallus gallus). The highly conserved residues are labeled with red triangles. (E, F) Structure superposition of the gMCM8 NTD/ mtMCM NTD (gMCM8, greencyan; mtMCM, PDB: 1LTL, grey). The ACL is highlighted by purple (gMCM8) (E). Structure-based alignment of MCM from different species are shown below the structures and key residues are labeled with red triangles (F). (G) Representative autoradiograph of the DNA unwinding reaction conducted using a 32P-labeled ssDNA oligo annealed to the M13mp18 as DNA substrate in the presence of HROB with purified MCM8/9 or mutants as indicated. (H) Graphical representation of the percentage of DNA unwinding in reactions conducted as in (G). The mean±SD of three independent experiments is presented. Statistical analysis was conducted using one-way ANOVA (**p<0.01, ***p<0.001).

Analysis of the helicase activities and chemoresistance exhibited by the N-C linkers.

(A) Representative autoradiograph of the DNA unwinding reaction conducted using a 32P-labeled ssDNA oligo annealed to the M13mp18 as DNA substrate in the presence or absence of HROB with purified MCM8/9, MCM8 N-C linker mutant (MCM8 Δ369-377) or MCM9 N-C linker mutant (MCM9Δ283-287), either alone or in combination. D-boiled DNA substrate control. (B) Graphical representation of the percentage of DNA unwinding in reactions conducted as in (A). The mean±SD of three independent experiments is presented. Statistical analysis was conducted using one-way ANOVA (****p<0.0001). (C) Western blot to detect MCM8 in DT40 control cells or in an MCM8 KO clone reconstituted with MCM8 WT or MCM8_Δ369-377 mutant. Actin is shown as a loading control. C, control cells; Δ369-377, MCM8_Δ369-377. (D) Survival analysis in DT40 control cell, MCM8 KO cell or cells reconstituted with MCM8 WT or MCM8 N-C linker mutant upon treatment with cisplatin. Cell survival is expressed as a percentage of an untreated control. The mean±SD of three independent experiments is presented. Statistical analysis was conducted on data points at four distinct cisplatin concentrations (200, 400, 600, 800 nM) using Student’s t-test (****p<0.0001, at all four concentrations analyzed). (E) Detection by western blot of MCM9 in DT40 control cells or in an MCM9 KO clone reconstituted with MCM9 WT or MCM9_Δ283-287 mutant. Actin is shown as a loading control. C, control cells; Δ283-287, MCM9_Δ283-287. (F) Survival analysis in DT40 control cell, MCM9 KO cell or cells reconstituted with MCM9 WT or MCM9 N-C linker mutant upon treatment with cisplatin. Cell survival is represented as in (D) and statistical analysis was conducted as in (D) (****p<0.0001, at all four concentrations analyzed).