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AP-MS of replisomes from S. cerevisiae in S phase and after unscheduled G1 replication

ABSTRACT: Interfering with once-per-cell-cycle regulation of DNA replication initiation generates genome instability through over-replication and has been linked to early stages of cancer development. Here, we engineered different systems in budding yeast to induce over-replication in G1 and investigated its characteristics and consequences within the same cell cycle. To study replisome composition, we immunopurified replisomes via a GFP-tag on the Psf2 subunit of the GINS complex from S phase in the presence and absence of hydroxyurea and after unscheduled replication initiation in G1.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Michael Wierer

LAB HEAD: Matthias Mann

PROVIDER: PXD028308 | Pride | 2022-11-09

REPOSITORIES: Pride

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			Action	DRS
	20190224_QX3_MiWi_ST00_replisome_G1-S_C_1.raw	Raw
	20190224_QX3_MiWi_ST00_replisome_G1-S_C_2.raw	Raw
	20190224_QX3_MiWi_ST00_replisome_G1-S_C_3.raw	Raw
	20190224_QX3_MiWi_ST00_replisome_G1-S_Psf2_1.raw	Raw
	20190224_QX3_MiWi_ST00_replisome_G1-S_Psf2_2.raw	Raw

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AP-MS of replisomes from S. cerevisiae in S phase and after unscheduled G1 replication

Project description:Interfering with once-per-cell-cycle regulation of DNA replication initiation generates genome instability through over-replication and has been linked to early stages of cancer development. Here, we engineered different systems in budding yeast to induce over-replication in G1 and investigated its characteristics and consequences within the same cell cycle. To study replisome composition, we immunopurified replisomes via a GFP-tag on the Psf2 subunit of the GINS complex from S phase in the presence and absence of hydroxyurea and after unscheduled replication initiation in G1.

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Project description:DNA replicates once per cell cycle. Interfering with the regulation of DNA replication initiation generates genome instability through over-replication and has been linked to early stages of cancer development. Here, we engineered genetic systems in budding yeast to induce unscheduled replication in the G1-phase of the cell cycle. Unscheduled G1 replication initiated at canonical S-phase origins across the genome. We quantified differences in replisomes in G1- and S-phase and identified firing factors, polymerase α, and histone supply as factors that limit replication outside S-phase. G1 replication per se did not trigger cellular checkpoints. Subsequent replication during S-phase, however, resulted in over-replication and led to chromosome breaks via head-to-tail replication fork collisions that are marked by chromosome-wide, strand-biased occurrence of RPA-bound single-stranded DNA. Low-level, sporadic induction of G1 replication induced an identical response, indicating findings from synthetic systems are applicable to naturally occurring scenarios of unscheduled replication initiation by G1/S deregulation.

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