Different nucleosomal landscapes at early and late replicating origins in Saccharomyces cerevisiae

Abstract

[ES]: Este trabajo aporta información relevante a la pregunta de cuáles son los factores que determinan el programa temporal de replicación en la levadura de gemación. Por primera vez se ha determinado que los orígenes de replicación tempranos y tardíos presentan distintos patrones nucleosomales, que están a su vez en estrecha relación con el contexto genómico. Además, se ha analizado la contribución de la acetilación de histonas en el establecimiento de la estructura de la cromatina en orígenes de replicación y se ha comprobado que aumentos de acetilación de histonas en los orígenes correlacionan con remodelamiento de la cromatina, dando lugar a una estructura más accesible. En conjunto, estos datos ayudan a entender la interacción entre diferentes factores que influyen en el tiempo de replicación de los orígenes en S. cerevisiae, como son la estructura de la cromatina, el contexto cromosómico y modificaciones epigenéticas.

[EN]: It has long been assumed that cells would not enter mitosis until chromosome replication is completed. This concept derives mainly from studies in which the progression of replication forks was halted by chemical (MMS, HU) or enzymatic impediments (e.g. ligase mutants). This causes exposure of long stretches of singlestranded DNA followed by activation of the DNA damage or replication checkpoint pathways that delay mitotic entry until the problem is fixed. In this work we tested in yeast whether cells that have not yet finished DNA replication, but in which fork progression is normal, can enter mitosis and become genetically unstable. To extend S phase without causing replication stress we used cdc6-1 and cdt1-21 ts mutants, which form fewer pre-replication complexes (pre-RCs) at semi-permissive temperatures. We showed using DNA combing that the density of active origins dropped proportionally to the rising temperature, along with a longer S phase and increased DNA damage response (Rad52 recombination foci, phospho-Rad53). Interestingly, DDR activation always took place 60 min after G1 release, the time at which cells normally enter mitosis. Crucially, we showed that DNA damage in cells containing under-replicated DNA was dependent on mitotic CDK and Cdc5/Plk1 kinase activities, as well as on phosphorylation of a replication fork protein. Our results demonstrate that i) cells can enter mitosis with underreplicated DNA, undetected by checkpoints, ii) mitotic entry is responsible for DNA damage or fork collapse in cells that replicate late, iii) completion of DNA synthesis occurs at least in part through illegitimate recombination-dependent mechanisms. Indeed Rad52 foci persist and anaphase is delayed for a long time in these cells, as expected for one end homology-driven repair. Accordingly, gross chromosome rearrangement (GCR) rates are very high in cdc6-1 and cdt1-21 mutants transiently grown at semi-permissive temperature. Our observations are reminiscent of the premature chromosome condensation phenotype caused by fusion of mitotic cells to S-phase nuclei. This work challenges the dogma that cells cannot enter mitosis before S phase is finished. It also provided a mechanism for the breakage of latereplicating common fragile sites (CFS) as well as a conceptual framework to explain the high chromosomal instability of cancer cells with altered replication programmes.