Cdc7-Drf1 is a developmentally regulated protein kinase required for the initiation of vertebrate DNA replication

Abstract

Cdc7, a protein kinase required for the initiation of eukaryotic DNA replication, is activated by a regulatory subunit, Dbf4. A second activator of Cdc7 called Drf1 exists in vertebrates, but its function is unknown. Here, we report that in Xenopus egg extracts, Cdc7-Drf1 is far more abundant than Cdc7-Dbf4, and removal of Drf1 but not Dbf4 severely inhibits phosphorylation of Mcm4 and DNA replication. After gastrulation, when the cell cycle acquires somatic characteristics, Drf1 levels decline sharply and Cdc7-Dbf4 becomes the more abundant kinase. These results identify Drf1 as a developmentally regulated, essential activator of Cdc7 in Xenopus.

Figure 1.

Cdc7-Drf1 is far more abundant than Cdc7-Dbf4 in Xenopus egg extracts. (A) Characterization of anti-Drf1 and Dbf4 antibodies: 0.2 μL LSS (lane 1), 0.2 μL λ-protein phosphatase-treated LSS (lane 2), 0.04 μL NPE (lane 3), and 0.04 μL λ-protein phosphatase-treated NPE (lane 4) were separated by SDS-PAGE and probed with anti-Drf1 (top panel) or Dbf4 (bottom panel) antibodies along-side 500 pg of recombinant Drf1 (6xHis and StrepII-tagged) (lane 5, top panel) or 20 pg recombinant Dbf4 (6xHis-tagged) (lane 5, bottom panel). Asterisks indicate cross-reacting bands. (B,C) Cdc7 complexes were immunoprecipitated from LSS by control (lane 1), anti-Cdc7 (lane 2), anti-Dbf4 (lane 3), or anti-Drf1 (lane 4) antibodies, and the supernatant (B) and immunoprecipitate (C) fractions were probed with antibodies against Drf1 (top panels), Dbf4 (middle panels), and Cdc7 (bottom panels). Cross-reacting bands (^*) serve as loading controls. In B and later figures involving Dbf4 blotting of LSS, extracts were treated with phosphatase due to a cross-reacting band migrating with phosphorylated Dbf4.

Figure 2.

Drf1 is required for DNA replication. Sperm chromatin was incubated in mock-depleted LSS supplemented with buffer (open squares) or 40 nM rCdc7-Drf1 (open circles), or Drf1-depleted LSS supplemented with buffer (filled squares) or 40 nM rCdc7-Drf1 (filled circles).

Figure 3.

Cdc7-Drf1 is required for initiation of DNA replication. (A) Drf1 is required for origin unwinding. pBluescriptII was incubated in mock-depleted (lane 1) or Drf1-depleted (lane 2) HSS at 40 ng/μL concentration for 30 min and isolated, or further incubated in mock-depleted (lanes 3-8) or Drf1-depleted (lanes 9-14) NPE containing 50 μg/mL aphidicolin and buffer (lanes 3-5,9-11) or 500 nM rCdc7-Drf1 (lanes 6-8,12-14). At the indicated times, the plasmid topology was analyzed by agarose gel electrophoresis in the presence of 1.8 μM chloroquine. (B) Requirement of Drf1 for Mcm4 phosphorylation and Cdc45 loading. Sperm chromatin was incubated in mock-depleted (lanes 1-4) or Drf1-depleted (lanes 5-8) LSS containing buffer (lanes 1,2,5,6) or 50 nM rCdc7-Drf1 (lanes 3,4,7,8) and recovered through a sucrose cushion at the indicated time points. Bound proteins were eluted and analyzed by Western blotting using the indicated antibodies. (^*) Cross-reacting band.

Figure 4.

Dbf4 is not required for DNA replication in Xenopus egg extracts. (A) The indicated amounts of mock-depleted (lanes 1-6) or Dbf4-depleted (lanes 7,8) LSS treated with λ-protein phosphatase were separated on SDS-PAGE and probed with anti-Dbf4 antibody; 100% = 1 μL. Cross-reacting bands (^*) serve as loading controls. (B) Replication efficiency in the mock-depleted (open squares) or Dbf4-depleted (filled squares) LSS shown in A. (C) Sperm chromatin was incubated in mock-depleted (lane 1) or Dbf4-depleted (lane 2) LSS in the presence of 50 μg/mL aphidicolin and isolated after 45 min. Western blotting for the indicated proteins is presented. (^*) Cross-reacting band. In D, 0.2 μL each of mock-depleted (lane 1), Drf1-depleted (lane 2), or Drf1/Dbf4-depleted (lane 3) LSS was treated with λ-protein phosphatase, separated by SDS-PAGE, and probed for the indicated proteins. (^*) Cross-reacting band. (E) Replication efficiency in mock-depleted (open squares), Drf1-depleted (filled squares), and Drf1- and Dbf4-depleted (filled triangles) LSS shown in D.(F) Replication efficiency in LSS that was mock-depleted (open squares), Drf1-depleted (filled squares), mock-depleted and supplemented with 1 μM recombinant Cdc7-Dbf4 (open circles), and Drf1-depleted and supplemented with 1 μM recombinant Cdc7-Dbf4 (filled circles).

Figure 5.

Developmental regulation of Drf1 and Dbf4. (A) Unfertilized Xenopus eggs (lane 1) or in vitro fertilized Xenopus embryos (lanes 2-13) were isolated at the indicated time points and staged according to Nieuwkoop and Faber (1967). Cdc7 complexes were immunoprecipitated from embryo lysates with anti-Cdc7 antibody and probed for Drf1 (top and second panels), Dbf4 (third and fouth panels), and Cdc7 (bottom panel). (B) Stage 3 in vitro fertilized Xenopus embryos (lanes 1-4) or stage 11 embryos (lanes 5-8) were collected and used for immunoprecipitation with control (lanes 1,5), anti-Cdc7 (lanes 2,6), anti-Dbf4 (lanes 3,7), and anti-Drf1 (lanes 4,8) antibodies. Western blotting for the indicated proteins is presented. An asterisk shows the IgG heavy chain, which reacts with the secondary antibody.