Understanding protein dynamics is crucial in order to elucidate protein function and interactions. Advances in modern microscopy facilitate the exploration of the mobility of fluorescently tagged proteins within living cells. Fluorescence recovery after photobleaching (FRAP) is an increasingly popular functional live-cell imaging technique which enables the study of the dynamic properties of proteins at a single-cell level. As an increasing number of labs generate FRAP datasets, there is a need for fast, interactive and user-friendly applications that analyze the resulting data. Here we present easyFRAP-web, a web application that simplifies the qualitative and quantitative analysis of FRAP datasets. EasyFRAP-web permits quick analysis of FRAP datasets through an intuitive web interface with interconnected analysis steps (experimental data assessment, different types of normalization and estimation of curve-derived quantitative parameters). In addition, easyFRAP-web provides dynamic and interactive data visualization and data and figure export for further analysis after every step. We test easyFRAP-web by analyzing FRAP datasets capturing the mobility of the cell cycle regulator Cdt2 in the presence and absence of DNA damage in cultured cells. We show that easyFRAP-web yields results consistent with previous studies and highlights cell-to-cell heterogeneity in the estimated kinetic parameters. EasyFRAP-web is platform-independent and is freely accessible at: https://easyfrap.vmnet.upatras.gr/.

Cell cycle checkpoints secure ordered progression from one cell cycle phase to the next. They are important to signal cell stress and DNA lesions and to stop cell cycle progression when severe problems occur. Recent work suggests, however, that the cell cycle control machinery responds in more subtle and sophisticated ways when cells are faced with naturally occurring challenges, such as replication impediments associated with endogenous replication stress. Instead of following a stop and go approach, cells use fine-tuned deceleration and brake release mechanisms under the control of ataxia telangiectasia and Rad3-related protein kinase (ATR) and checkpoint kinase 1 (CHK1) to more flexibly adapt their cell cycle program to changing conditions. We highlight emerging examples of such intrinsic cell cycle checkpoint regulation and discuss their physiological and clinical relevance. From Stop and Go Cell Cycle Decisions to a Continuum of Deceleration and Brake Release MechanismsCell cycle checkpoints (see Glossary) are control mechanisms that ensure proper and ordered progression of cells through the cell cycle [1]. Genome integrity maintenance is among the key tasks of cell cycle checkpoint control, and DNA damage checkpoints have thus evolved to trigger cell cycle arrest in response to genomic lesions and allow time for repair [2]. The cell cycle checkpoint model, in agreement with the general meaning of the term checkpoint, implies certain criteria, which have to be met by a cell to continue its progression through the cell cycle (the 'go' decision) and which, if they are not fulfilled, lead to transient or permanent cell cycle arrest (the 'stop' decision). Among the major cell cycle checkpoints are the G1/S checkpoint, the G2/M checkpoint, and the spindle checkpoint in mitosis. By analogy to 'the hammer and the dance' metaphor introduced early in 2020 by Tomas Pueyo to emphasize the importance of acting quickly and forcefully to contain the global health threat posed by SARS-CoV-2 and later ease and adapt the measures to continually manage and control COVID-19 ('Coronavirus: the hammer and the dance', published online March 19, 2020; https://medium.com/@tomaspueyo/coronavirus-the-hammerand-the-dance-be9337092b56), we can consider full checkpoint activation to halt cell cycle progression as 'the hammer', which can be an effective means to minimize further damage in the face of severe threats to genome integrity. However, rather than relying exclusively on binary stop and go decisions, a picture is starting to emerge in which the cell cycle control machinery uses fine-tuned brakes and brake release mechanisms [3], thereby temporarily decelerating certain processes while continuing others, to globally balance genome surveillance with cell cycle progression ('the dance'). Thus, cell cycle arrest as an extreme measure seems reserved for severe events that threaten genome integrity and cell survival, while more moderate and adaptable measures are commonly used by cells to deal with controllable genomic lesions and repl...

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The C-terminal end of Cdt2 contains a PIP box for binding to PCNA to promote CRL4Cdt2 function, creating a new paradigm where the substrate receptor and substrates bind to a common multivalent docking platform for ubiquitination.

The paradigm that checkpoints halt cell cycle progression for genome repair has been challenged by the recent discovery of heritable DNA lesions escaping checkpoint control. How such inherited lesions affect genome function and integrity is not well understood. Here, we identify a new class of heritable DNA lesions, which is marked by replication protein A (RPA), a protein primarily known for shielding single-stranded DNA in S/G2. We demonstrate that post-mitotic RPA foci occur at low frequency during unperturbed cell cycle progression, originate from the previous cell cycle, and are exacerbated upon replication stress. RPA-marked inherited ssDNA lesions are found at telomeres, particularly of ALT-positive cancer cells. We reveal that RPA protects these replication remnants in G1 to allow for post-mitotic DNA synthesis (post-MiDAS). Given that ALT-positive cancer cells exhibit high levels of replication stress and telomere fragility, targeting post-MiDAS might be a new therapeutic opportunity.