Genome-wide hierarchy of replication origin usage in Saccharomyces cerevisiae

Abstract

Replication origins in a genome are inherently different in their base sequence and in their response to temporal and cell cycle regulation signals for DNA replication. To investigate the chromosomal determinants that influence the efficiency of initiation of DNA replication genome-wide, we made use of a reverse strategy originally used for the isolation of replication initiation mutants in Saccharomyces cerevisiae. In yeast, replication origins isolated from chromosomes support the autonomous replication of plasmids. These replication origins, whether in the context of a chromosome or a plasmid, will initiate efficiently in wild-type cells but show a dramatically contrasted efficiency of activation in mutants defective in the early steps of replication initiation. Serial passages of a genomic library of autonomously replicating sequences (ARSs) in such a mutant allowed us to select for constitutively active ARSs. We found a hierarchy of preferential initiation of ARSs that correlates with local transcription patterns. This preferential usage is enhanced in mutants defective in the assembly of the prereplication complex (pre-RC) but not in mutants defective in the activation of the pre-RC. Our findings are consistent with an interference of local transcription with the assembly of the pre-RC at a majority of replication origins.

Figure 1. Serial Passages of a Plasmid Library of Genomic DNA in mcm1–1 Cells to Enrich for Stable Plasmids Containing A-Type ARSs

Figure 2. YCp121 Is More Stable than YCp1 in mcm1–1 Cells

Cells (wild-type or mutant) containing YCp121 or YCp1 were mixed (A). YCp121 and YCp1 were mixed and transformed into wild-type or mcm1–1 cells (B). Colonies on selective medium were pooled and used to inoculate a culture in selective media. The culture was grown to saturation (one passage) and used to inoculate a fresh culture of selective media. This process was repeated two or three times. The relative abundance of YCp1 containing ARS1 and YCp121 containing ARS121 was measured at the end of a passage.

Figure 3. Characterization of A-Type and B-Type ARSs

(A) Effect of insert size on plasmid stability. Loss rates of plasmids per cell division in mcm1–1 cells plotted against insert size. Only inserts between 3,800 and 9,300 bp were analyzed further. (B) Stability of plasmids was measured in mutant (black diamonds) and wild-type (white squares) cells.

Figure 4. Effect of Local Transcription Pattern on Plasmid Stability

Three sets of deletion plasmid constructs and their corresponding loss rates in mcm1–1 cells are shown.

Figure 5. Comparing the Effect of mcm1–1 on A-Type and B-Type ARSs with Those of Pre-RC Assembly (cdc6, orc2, and mcm3) and Activation (cdc45 and dbf4) Mutants as Well as mcm10 in a mcm Assay

The asterisk indicates rate was too high to be measured. ARS1010, ARS905, and ARS1605 are B-type ARSs. ARS1009, ARS202, and ARS121 are A-type ARSs.