Replisome loading reduces chromatin motion independent of DNA synthesis

Abstract

Chromatin has been shown to undergo diffusional motion, which is affected during gene transcription by RNA polymerase activity. However, the relationship between chromatin mobility and other genomic processes remains unclear. Hence, we set out to label the DNA directly in a sequence unbiased manner and followed labeled chromatin dynamics in interphase human cells expressing GFP-tagged PCNA, a cell cycle marker and core component of the DNA replication machinery. We detected decreased chromatin mobility during the S-phase compared to G1 and G2 phases using automated particle tracking. To gain insight into the dynamical organization of the genome during DNA replication, we determined labeled chromatin domain sizes and analyzed their motion in replicating cells. By correlating chromatin mobility proximal to the active sites of DNA synthesis, we showed that chromatin motion was locally constrained at the sites of DNA replication. Furthermore, inhibiting DNA synthesis led to increased loading of DNA polymerases. This was accompanied by accumulation of the single-stranded DNA binding protein on the chromatin and activation of DNA helicases further restricting local chromatin motion. We, therefore, propose that it is the loading of replisomes but not their catalytic activity that reduces the dynamics of replicating chromatin segments in the S-phase as well as their accessibility and probability of interactions with other genomic regions. – Direct and sequence unbiased labeling of DNA genome-wide – DNA labeled chromatin is more mobile in G1/G2 relative to the S-phase – Restriction of chromatin motion occurs proximal to sites of DNA replication – Loading of replisomes, even in the absence of processive DNA synthesis, restricts chromatin motion

DNA choreography: correlating mobility and organization of DNA across different resolutions from loops to chromosomes

“…G1397-100 ml, Th.Geyer GmbH, Germany) in a humidified atmosphere with 5% CO 2 at 37 °C. Previously published experiments confirmed that the transgenic gene product colocalized with the endogenous protein and was present at sites of active replication (Chagin et al 2016 ; Pabba et al 2023 ). The culture medium was changed every day and cells were split every 2 days.…”

“…All cells used were tested and negative for mycoplasma. Human cervical cancer cell line HeLa Kyoto (Erfle et al 2007 ) and HeLa K cells expressing GFP-PCNA (Chagin et al 2016 ; Pabba et al 2023 ) fusion protein were used to monitor cell cycle progression. All cells were maintained in Dulbecco’s modified Eagle medium (DMEM) (Cat.…”

“…Notably, chromatin, the blend of DNA and proteins that constitutes chromosomes, is far from a static entity but rather exhibits motion within the cell nucleus. This motion dynamically interplays with (patho)physiological cellular processes such as DNA transcription (Nozaki et al 2017 ; Tortora et al 2020 ; Miron et al 2020 ; Laghmach et al 2021 ; Mach et al 2022 ), DNA replication (Heun et al 2001 ; Levi et al 2005 ; Levi and Gratton 2008 ; Zidovska et al 2013 ; Tortora et al 2020 ; Gabriele et al 2022 ; Pabba et al 2023 ), DNA repair (Miné-Hattab et al 2017 ), and cellular senescence (Shaban and Gasser 2023 ) and diseases (Li et al 2022 ). For instance, during transcriptional activation or repression, specific genetic loci undergo conformational changes that involve alterations in chromatin structure and motion (Germier et al 2017 ; Shaban et al 2018 ; Shaban and Suter 2022 ).…”

“…For instance, during transcriptional activation or repression, specific genetic loci undergo conformational changes that involve alterations in chromatin structure and motion (Germier et al 2017 ; Shaban et al 2018 ; Shaban and Suter 2022 ). Furthermore, the dynamic nature of chromatin is indispensable for the successful execution of DNA replication and repair processes by facilitating the access to the DNA segments undergoing replication or repair (Zidovska et al 2013 ; Miné‑Hattab et al 2017 ; Ma et al 2019 ; Ochs et al 2019 ; Pabba et al 2023 ). Previous studies have shown that chromatin motion plays an important role in maintaining interactions between topologically associated domains (TADs) for an average of 10 min to facilitate gene expression and this was shown to be enabled by cohesin and CTCF (Mach et al 2022 ).…”

Timeless–Tipin interactions with MCM and RPA mediate DNA replication stress response

“…An accumulation of RPA in the vicinity of the stalled replication fork is one of the replication stress hallmarks. For an easier evaluation of RPA accumulation upon stress, we used a HeLa cell line ectopically expressing GFP-RPA ( Pabba et al, 2023 ). Figure 3A (right panel) shows a large accumulation of RPA after HU/APH treatment, which confirms a successful stress induction.…”