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. 2015 Jun 16;34(15):2096–2110. doi: 10.15252/embj.201488016

Figure 6.

Figure 6

And-1 promotes the association of Claspin with ssDNA

  1. And-1 binds to ssDNA in a dose-dependent manner. Immobilized 80-mer ssDNA was incubated in reactions with buffer, BSA (200 or 400 ng), or recombinant And-1 (200 or 400 ng) for 30 min at room temperature. The beads were washed, and bound proteins were resolved by SDS–PAGE and immunoblotted for the indicated proteins.
  2. And-1 binds preferably to ssDNA rather than dsDNA. ssDNA was annealed with buffer or its complementary oligonucleotide before immobilized onto magnetic beads. Immobilized ssDNA or dsDNA was incubated with recombinant And-1. The beads were washed and treated as in (A).
  3. And-1 promotes the association of Claspin with ssDNA. Immobilized 80-mer ssDNA was incubated in reactions with BSA or And-1 for 30 min at room temperature followed by incubation with recombinant Claspin at 4°C for 2 h. Beads were washed and treated as in (A). Recombinant Claspin proteins purified from 293T cells (left) or insect cells (right).
  4. Phosphorylation of And-1 at T826 enhances the association of Claspin with ssDNA. And-1 or And-1(T826) recombinant proteins were purified from 293T cells treated with or without HU, and were first incubated with ssDNA and then with recombinant FLAG-Claspin proteins as described in (C).
  5. And-1 co-localizes with ssDNA. HCT116 cells were labeled with BrdU 10 μM for 48 h, covered with a 5-μm polycarbonate isopore membrane filter, and subjected to 100 J/m2 UV irradiation. Harvested cells were immunostained for And-1 and BrdU without DNA denaturation. The co-localization of And-1 with BrdU was observed in 97% of BrdU foci-positive cells. Data are from three independent experiments. Scale bar, 5 μm.
  6. Endogenous And-1 is associated with replication forks. Asynchronous 293T cells were harvested for iPOND assay (see Materials and Methods for detail).
  7. And-1 is a bona fide replication fork protein. U2OS cell line constitutionally expressing FLAG-And-1 was labeled with EdU, or chased with regular growth medium for 2 h after EdU labeling to be used as chromatin pull-down control.
  8. And-1 is accumulated on stalled replication forks. 293T cells transfected with wild-type or T826A mutant FLAG-And-1 plasmids were treated with 10 mM HU for 2 h before harvested for iPOND assay. The intensity of Western blots was quantified with ImageJ software and indicated below the blots. The average relative intensity of bands is from three independent experiments.

Source data are available online for this figure.