Cdc7 is required throughout the yeast S phase to activate replication origins

Abstract

The long-standing conclusion that the Cdc7 kinase of Saccharomyces cerevisiae is required only to trigger S phase has been challenged by recent data that suggests it acts directly on individual replication origins. We tested the possibility that early- and late-activated origins have different requirements for Cdc7 activity. Cells carrying a cdc7(ts) allele were first arrested in G1 at the cdc7 block by incubation at 37 degrees C, and then were allowed to enter S phase by brief incubation at 23 degrees C. During the S phase, after return to 37 degrees C, early-firing replication origins were activated, but late origins failed to fire. Similarly, a plasmid with a late-activated origin was defective in replication. As a consequence of the origin activation defect, duplication of chromosomal sequences that are normally replicated from late origins was greatly delayed. Early-replicating regions of the genome duplicated at approximately their normal time. The requirements of early and late origins for Cdc7 appear to be temporally rather than quantitatively different, as reducing overall levels of Cdc7 by growth at semi-permissive temperature reduced activation at early and late origins approximately equally. Our results show that Cdc7 activates early and late origins separately, with late origins requiring the activity later in S phase to permit replication initiation.

Figure 1

Scheme of experimental procedures. A,B,and C show time lines representing the course of G₁ and S phase under different experimental conditions. (A) Entry into and passage through a normal S phase. Before entry into S phase, cells transmit the α-factor arrest point and cdc7 arrest point, indicated by arrows. The rectangular box represents S phase. Replication origin activation events are drawn as shaded circles, with lighter shading indicating early-activated replication origins and darker shading for later origins. (B) Scheme of the experimental approach of Hartwell (1973) employing a cdc7-4 mutant. Vertical offset in lines represents temperature shift. Broken circles and question mark indicate replication origins that may not have been active under the particular experimental conditions. Although the temperature shift is drawn as having taken place two-fifths of the way through S phase, it should be noted that this is arbitrary; it was not possible to determine the time in S phase at which cells were shifted. (C) Illustration of experimental approach taken in this study, employing a cdc7-1 mutant. Broken circles as in B. See text for further explanation.

Figure 2

Two-dimensional gel analysis of early and late replication origin activation during S phase after a pulse of Cdc7 activity. (A) The cartoon depicts the structure of replication intermediates in the arcs of Y and bubble-shaped molecules. (B,D,F) Cells released from the cdc7 block and kept at 23°C (23°C S phase). (C,E,G) Cells released from a cdc7 block, and returned to 37°C after 4.5 min at 23°C (37°C S phase). B and C were probed to detect a fragment containing the early replication origin ARS306. D and E show a fragment containing the late origin ARS1412. F and G were probed for a restriction fragment (3LT) close to the left end of chromosome III.

Figure 3

Replication kinetics after a pulse of Cdc7 activity. (A) Cells released at 23°C from a cdc block and returned to 37°C after 3.5 min at 23°C. (B) Control cells released from the cdc7 block and kept at 23°C. Replication kinetics of two early-replicating standard fragments, containing ARS305 (solid line with open rectangles) and ARS1 (broken line with filled ovals), and two late-replicating standard fragments, R11 (broken line with filled rectangles) and a fragment containing ARS1412 (solid line with open triangles) are shown.

Figure 4

Replication kinetics of an early-duplicating plasmid containing ARS306. Kinetics of replication of the plasmid p306.10 (solid line with open triangles), of early chromosomal fragments containing ARS305 (dotted line with open squares) and ARS1 (dashed line with solid circles), and the late chromosomal fragment R11 (solid line with solid squares) are shown.

Figure 5

Replication kinetics of early and late plasmids after a pulse of Cdc7 activity. Cultures held at the cdc7 block were given a 7.5-min pulse at 23°C, then returned to 37°C. (A) Cells containing the early-replicating plasmid p306.10. (B) Cells containing the late-replicating plasmid p12. Plasmid replication levels in each culture are indicated by solid triangles. The replication curves of chromosomal early marker ARS305 (open circles) and late marker R11 (open squares) in the two cultures are also shown.

Figure 6

Two-dimensional gel analysis of early and late replication origin activation at semipermissive temperatures for the cdc7-1 allele. (Top) Blots probed for replication intermediates of a fragment containing early origin ARS306. (Bottom) Blots probed for the late origin ARS1412. Left panels show DNA from an asynchromous culture grown at permissive temperature (23°C). Center and right panels show blots made with DNA from cultures grown at semipermissive temperatures (26.5°C and 27°C).