Fig. 3: Factors limiting unscheduled G1 replication include availability of firing factors and histones.

a Origin licensing does not limit levels of unscheduled G1 replication. Cells were kept arrested in G1 and induced to replicate DNA using the CDK/DDK-bypass system with or without additional galactose-inducible expression of licensing factor Cdc6. (left) SYTOX green-stained total DNA after induction of G1 replication measured by flow cytometry at indicated timepoints. (right) Quantification of total DNA data in left panel by approximation of a bimodal distribution and calculating means of individual normal distributions. The average mean from 5 fits per timepoint is shown together with a linear regression. Data are representative of n = 2 biological replicates. b CDK/DDK bypass for unscheduled replication in G1 is limited by the availability of initiation factors and efficient bypass of CDK-control. Experiment and analysis as in (a) but cells additionally expressed high levels of firing factors Sld3/Sld7 as well as helicase component Cdc45 either as a wild-type or as a JET1-mutant, which bypasses CDK-regulation of Sld3. Data are representative of n = 2 biological replicates. c Activation of ribonucleotide reductase (RNR) does not lead to an increase of G1 replication. Experiment and analysis as in (a) but cells additionally expressed the indicated RNR1 alleles or lacked negative RNR regulators Sml1 and Dif1. Data are representative of n = 2 biological replicates. d Increasing histone availability through transcription factor Spt21 increases unscheduled replication in G1. Experiment and analysis as in (a) but cells were additionally expressing high-levels of transcription factor Spt21 which regulates transcription of histone genes and is normally degraded in G1. Data are representative of n = 2 biological replicates.