Journal: Nature Communications
Article Title: Unscheduled DNA replication in G1 causes genome instability and damage signatures indicative of replication collisions
doi: 10.1038/s41467-022-34379-2
Figure Lengend Snippet: a Summary of engineered genetic changes that allow to bypass cell cycle control of DNA replication initiation and induce unscheduled replication in G1. Indicated proteins and variants are expressed from a galactose-inducible pGAL1-10 promoter. Experimental setup for G1 replication involves G1 cell cycle arrest using α-factor in raffinose medium, followed by induction of G1 replication by addition of galactose. Expression of Dpb11 and Sld2-T84D allows for bypass of CDK controls, additional expression of Dbf4 allows for bypass of DDK controls. Pictograms indicate genetic bypass in all figures. b Bypassing CDK control and CDK/DDK control generates different levels of unscheduled replication in G1. Cells were arrested in G1 and replication was induced by adding galactose. EdU (100 µM) was added to the G1-arrested cells right after induction of replication. Cells were harvested at indicated timepoints after replication induction and EdU-containing DNA was labeled with Cy5. Total DNA content (stained by SYTOX green) and newly synthesized DNA (EdU-Cy5-labeled) were measured by flow cytometry. Data are representative of n = 3 biological replicates. c Unscheduled replication in G1 after bypass of CDK or CDK/DDK control occurs genome-wide. Experiment as in ( b ), but EdU-labeled DNA as a proxy for DNA synthesis was isolated after 3 h of G1 replication and sequenced. Sequencing reads were mapped to the S. cerevisiae genome and normalized for input total DNA. Data are representative of n = 2 biological replicates. d and e Unscheduled G1 replication initiates at canonical replication origins. Experiment as in ( b )/( c ), but 60 mM hydroxyurea (HU) was added to the medium when replication was induced. d G1 replication initiates at early-firing replication origins (autonomous replicating sequences (ARS)). Input-normalized coverage of 60 kb windows shows EdU-labeled replication products around ARS after 3 h replication in G1 at ARS firing either early (blue) or late (green) in S-phase. (top) Summarizing profile plots of mean coverage (dark) ± SE (light) at the ARSs ± 30 Kb. (bottom) Heatmaps with 2 Kb bin size, each row corresponds to an individual ARS. Data are representative of n = 2 biological replicates. e Representative example of input-normalized traces for EdU-containing DNA spanning the entire chromosome 4. The scale of the y- axis (log) is given in the top-left corner. Dotted lines indicate early-replicating ARSs.
Article Snippet: One half was subjected to a click chemistry reaction with disulfo-Cy5-picolyl-azide (Jena Bioscience CLK-1177) for 1 h, whereas the other half was kept as a control.
Techniques: Expressing, Labeling, Staining, Synthesized, Flow Cytometry, Genome Wide, DNA Synthesis, Isolation, Sequencing
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