The B-subunit of DNA polymerase alpha-primase associates with the origin recognition complex for initiation of DNA replication

Abstract

The B-subunit (p70/Pol12p) of the DNA polymerase alpha-primase (Polalpha-primase) complex is thought to have a regulatory role in an early stage of S phase. We generated a panel of fission yeast thermosensitive mutants of the B-subunit (termed Spb70) to investigate its role in initiation of DNA replication by genetic and biochemical approaches. Here, we show that the fission yeast Spb70 genetically interacts and coprecipitates with origin recognition complex proteins Orp1/Orc1 and Orp2/Orc2 and primase coupling subunit Spp2/p58. A fraction of Spb70 associates with Orp2 on chromatin throughout the cell cycle independent of the other subunits of Polalpha-primase. Furthermore, primase Spp2/p58 subunit preferentially associates with the unphosphorylated Orp2, and the association requires Spb70. Mutations in orp2+ that abolish or mimic the Cdc2 phosphorylation of Orp2 suppress or exacerbate the thermosensitivity of the spb70 mutants, respectively, indicating that an unphosphorylated Orp2 promotes an Spb70-dependent replication event. Together, these results indicate that the chromatin-bound B-subunit in association with origin recognition complex mediates recruiting Polalpha-primase complex onto replication origins in G1 pre-Start through an interaction with primase Spp2/p58 subunit. Our results thus suggest a role for the recruited Polalpha-primase in the initiation of both leading and lagging strands at the replication origins.

FIG. 1.

Identification of the fission yeast B-subunit of DNA Polα-primase complex. (A) Expression of two-myc-six-His-tagged Spb70 in cells. The two-myc- and six-His-tagged spb70⁺ and untagged spb70⁺ strain were grown at 25°C in EMM to mid-log phase. Cells were harvested and whole-cell extracts (W.C.E.) were prepared as described in Materials and Methods. The expression of Polα/p180, Spp2/p58, and Spp1/p49 and absence of two-myc-six-His-tagged Spb70 in whole-cell extracts from cells containing spb70⁺ with no epitope tag (−) and expression of two-myc-six-His-tagged Spb70 in whole-cell extracts from cells containing the two-myc-six-His-tagged spb70⁺ (+) are shown by Western blotting with their respective antibodies and anti-myc antibody. CBB, the Coomassie brilliant blue staining of the Western blot membrane. (B) Spb70 physically associates with Polα/p180 and primase Spp1/p49 and Spp2/p58. Ni bead pull-down fractions of whole-cell extracts from the untagged spb70⁺ strain (−) and from the two-myc-six-His-tagged spb70⁺ strain (+) were blotted with their respective antibodies. (C) Schematic diagram of cell fractionation. (D) Spb70 localizes in both Triton X-100-soluble and -insoluble fractions. The strain containing two-myc-six-His-spb70⁺ and HA-orp1⁺ was grown at 25°C in EMM to mid-log phase. Cells were harvested, and Triton X-100-soluble and -insoluble (chromatin-enriched) fractions were prepared as described in Materials and Methods and outlined in panel C. HA-tagged Orp1 was used as the Triton X-100-insoluble protein control in chromatin-enriched fraction; two-myc-six-His-Spb70 and Spp2/p58 in each fraction was shown by Western blotting with their respective antibodies. As described in Materials and Methods, the intensity of each protein shown in Western blotting does not represent their molar quantities in cell extracts and Ni bead pull-downs due to the difference in reactivity of each antibody. CBB, the Coomassie brilliant blue staining of the immunoblot membrane.

FIG. 2.

Thermosensitive mutants of spb70⁺. (A) Thermosensitivity of spb70 mutants. Wild type (wt), cdc45^goa1, spb70-U01, spb70-U02, spb70-U03, and spb70-U04 were first cultured in liquid YES medium at 25°C. Equal numbers of log-phase cells were spotted on YES plates in threefold serial dilution and incubated at 25, 30, and 36°C for 3 days. (B) FACS profile of spb70-U02. cdc10-M17, spb70-U02, and cdc17-K42 were first grown at 25°C in YES medium followed by shifting to 36°C for 4 h. cdc10 and cdc17 were used as 1C and 2C DNA content standards, respectively. (C) Terminal phenotype of spb70-U02. Exponentially growing cells were shifted to 36°C for 4 h and stained with DAPI.

FIG. 3.

Genetic and physical characterizations of spb70 mutants. (A) Genetic interactions. Wild type (wt), single mutants spb70-U12, cdc45^goa1, and spp2-8, and double mutants spb70-U12 cdc45^goa1 and spb70-U12 spp2-8 were cultured in liquid YES medium at 25°C. Equal numbers of log-phase cells were spotted on YES plates in threefold serial dilution and incubated at 25 and 30°C for 3 days. (B) Terminal phenotypes. Single mutants spb70-U12 and spp2-8 and double mutant spb70-U12 spp2-8 were first grown in liquid YES medium at 25°C then shifted up to 36°C for 4 h. Cells were harvested and stained with DAPI and calcofluor. (C) Stability of Spb70 protein and interaction of Spb70 with other Polα-primase subunits. Wild-type two-myc-six-His-spb70⁺ and mutant two-myc-six-His-spb70-U03 cells were cultured at 25°C in EMM to mid-log phase and then shifted to 36°C. At the indicated time, cells were harvested and whole-cell extracts (W.C.E.) were prepared. The Spb70 protein levels in whole-cell extracts from spb70⁺ and spb70-U03 cells were detected by anti-myc antibody (W.C.E. panel). Spp2/p58 protein levels in whole-cell extracts from spb70⁺ and spb70-U03 cells after the shift to 36°C for 4 h used for the Ni bead pull-down are shown in the bottom panel. Ni bead pull-down fractions from wild-type two-myc-six-His-spb70⁺ and mutant two-myc-six-His-spb70-U03 whole-cell extracts after shifting the cells to 36°C for 4 h were analyzed by Western blotting with anti-Polα antibody, anti-HA for Spp2/p58, and anti-myc antibody for Spb70 for association of Polα/p180 and Spp2/p58 with Spb70 protein, respectively (Ni-beads panel). HA-tagged Spp2 proteins were immunoprecipitated from whole-cell extracts of the wild type containing two-myc-six-His-spb70⁺ and spp2⁺-three-HA and the mutant containing two-myc-six-His-spb70-U03 and spp2⁺-three-HA with anti-HA antibody. The immunoprecipitates were analyzed by Western blotting with anti-myc for Spb70 and anti-HA antibody for Spp2 (I.P Spp2 panel).

FIG. 4.

Spb70 genetically and physically interacts with ORC proteins. (A) Genetic interaction. Wild type (wt), orp1-4, spb70-U03, and double mutant spb70-U03 orp1-4 were cultured in liquid YES medium at 25°C. Equal numbers of log-phase cells were spotted on YES plates in threefold serial dilution and incubated at 25 and 30°C for 4 days. (B) Spb70 protein physically associates with Orp1/Orc1. HA-orp1^+/orc1 and two-myc-six-His-spb70⁺ HA-orp1⁺ strains were grown at 25°C in EMM to mid-log phase and whole-cell extracts (W.C.E.) were prepared from each strain. Presence of Spb70 and Orp1 in whole-cell extracts was detected by anti-myc and anti-HA antibody, respectively (W.C.E. panel). Ni bead pull-down fractions were Western blotted with anti-myc for Spb70 and anti-HA for Orp1 (Ni-beads panel). (C) Spb70 protein physically associates with Orp2/Orc2, and a mutation in spb70⁺ compromises the interaction. Wild-type cells containing two-myc-six-His-spb70⁺ and orp2⁺-three-FLAG and the mutant containing two-myc-six-His-spb70-U03 and orp2⁺-three-FLAG were cultured at 36°C. Ni bead pull-down fractions from each strain's chromatin fraction were Western blotted with anti-myc and anti-FLAG for Spb70 and Orp2, respectively. The Orp2 proteins in the chromatin fraction of wild-type and mutant cells used for the Ni bead pull-down experiment are shown in the bottom panel.

FIG. 5.

Spb70 associates with Orp2/Orc2 throughout the cell cycle. The cdc25-22 strain containing two-myc-six-His-spb70⁺ and orp2⁺-three-FLAG was grown at 25°C in EMM to mid-log phase and then shifted to 36°C to arrest at G₂ for 4 h. Cells were then released from G₂ arrest by shift-down to 25°C and harvested every 20 min after release from G₂ arrest. Chromatin fractions were prepared from each cell sample as described in Materials and Methods. Presence of Mcm6, Spb70, Orp2, and Spp2 in chromatin fraction was detected by Western blotting with anti-MCM6, anti-myc, anti-FLAG, and anti-Spp2 antibodies, respectively. (A) Cell cycle progression after the release from G₂ arrest. Percentage of cells in different stages of the cell cycle was shown as the septation index or mitotic index. (B) Proteins in chromatin-enriched fractions. Kinetics of the appearance of MCM6, Orp2, primase Spp2/p58, and Spb70 in the chromatin fraction after release from G₂ arrest were shown by Western blotting each protein with their respective antibody. CCB, the Coomassie brilliant blue staining of the Western blot membrane. (C) Coprecipitation of Spb70 and Orp2. Immunoprecipitates of Orp2 by anti-FLAG were Western blotted with anti-myc for Spb70 and anti-FLAG for Orp2.

FIG. 6.

Phosphorylation and unphosphorylation of Orp2 affects the temperature-sensitive growth of spb70 mutants. (A) Mutants orp2-T4A and orp2-T3D exert opposite effects on spb70-U05. Single mutants orp2-T4A, orp2-T3D, and spb70-U05 and double mutants spb70-U05 orp2-T4A and spb70-U05 orp2-T3D were cultured in liquid YES medium at 25°C. Equal numbers of log-phase cells from each strain were spotted on YES plates in threefold serial dilution and incubated at 25 and 35°C for 2 days. (B) orp2-T4A, but not orp2-T3D, suppresses the temperature sensitivity of spb70-U05 at 36.5°C. Thermosensitivity of double mutant spb70-U05 orp2-T4A was compared to single mutant spb70-U05 and double mutant spb70-U05 orp2-T3D after 3 days of incubation at 36.5°C. (C) Suppression of the temperature sensitivity of spb70-U12 by orp2-T4A. Single mutants orp2-T4A and spb70-U12 and double mutant spb70-U12 orp2-T4A were incubated at 30°C for 2 days.

FIG. 7.

Spp2/p58 interacts with Orp2, and the interaction requires Spb70. Wild-type cells containing orp2⁺-three-FLAG either with or without spp2⁺-three-HA and mutant spb70-U12 containing orp2⁺-three-FLAG and spp2⁺-three-HA were cultured in EMM to mid-log phase at 25°C and then shifted to 36°C for 3.5 h. Whole-cell extracts (W.C.E.) were prepared from each strain. Presence or absence of HA-tagged Spp2/p58 and FLAG-tagged Orp2 in whole-cell extracts was shown by Western blotting (W.C.E. panel). Spp2/p58 proteins were immunoprecipitated from each strain with anti-HA antibody, and the immunoprecipitates were Western blotted with anti-HA for Spp2/p58 and anti-FLAG for Orp2 (I.P. Spp2 panel). In whole-cell extracts, Orp2-FLAG protein was detected in whole-cell extracts from spb70⁺ strains either with or without spp2⁺-three-HA (lanes 1 and 2) and from mutant spb70-U12 (lane 3), while Spp2-HA was not detected in whole-cell extracts from spb70⁺ cells without spp2⁺-three-HA (lane 4) and only detected in whole-cell extracts from spb70⁺ and spb70-U12 strains containing spp2⁺-three-HA (lanes 5 and 6). In the anti-HA immunoprecipitates (I.P. Spp2 panel), Orp2-FLAG coprecipitated with Spp2/p58 from whole-cell extracts in the spb70⁺ strain containing spp2⁺-three-HA (Lane 8) but not from mutant spb70-U12 containing spp2⁺-three-HA (lane 9). Anti-HA immunoprecipitates from whole-cell extracts of the spb70⁺ strain without spp2⁺-three-HA had neither detectable Orp2-FLAG nor Spp2/p58-HA (lanes 7 and 10), and Spp2-HA was detected in immunoprecipitates from whole-cell extracts of spb70⁺ and spb70-U12 strains containing spp2⁺-three-HA (lanes 11 and 12).

FIG. 8.

Spp2/p58 coprecipitates with unphosphorylated Orp2/Orc2. The cdc25-22 strain containing two-myc-six-His-spb70⁺, orp2⁺-three-FLAG, and spp2⁺-three-HA was cultured at 25°C in EMM to mid-log phase and shifted to 36°C for 4 h to arrest at G₂. Cells were then released from G₂ arrest by shift to 25°C and harvested every 12 min. Chromatin fractions were prepared as described in Materials and Methods. (A) Cell cycle progression was monitored by septation index and mitotic index. (B) Kinetics of the appearance of MCM6, Orp2, Spp2/p58, and Spb70 in the chromatin fractions prepared from each time point were detected by Western blotting with their respective antibodies as described for Fig. 5B. (C) Coprecipitations of the unphosphorylated Orp2-FLAG with the HA-tagged Spp2/p58 were shown by Western blotting the anti-HA immunoprecipitates of Spp2 with anti-FLAG and anti-HA antibodies. A demonstration of phosphorylated and unphosphorylated Orp2 is shown next to the 0-min time point.