Deregulated origin licensing and rereplication promote genome instability and tumorigenesis by largely elusive mechanisms. Investigating the consequences of Early mitotic inhibitor 1 (Emi1) depletion in human cells, previously associated with rereplication, we show by DNA fiber labeling that origin reactivation occurs rapidly, well before accumulation of cells with >4N DNA, and is associated with checkpoint-blind ssDNA gaps and replication fork reversal. Massive RPA chromatin loading, formation of small chromosomal fragments, and checkpoint activation occur only later, once cells complete bulk DNA replication. We propose that deregulated origin firing leads to undetected discontinuities on newly replicated DNA, which ultimately cause breakage of rereplicating forks.

The mediators of the DNA damage response (DDR) are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK) complexes. We find that a specific G2/M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR–specific protein kinases.

Many important discoveries in cell cycle research have been made using cell-free extracts prepared from the eggs of the South African clawed frog Xenopus laevis. These extracts efficiently support the key nuclear functions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. The Xenopus cell-free system is therefore uniquely suited to the study of the mechanisms, dynamics and integration of cell cycle regulated processes at a biochemical level. Here, we describe methods currently in use in our laboratory for the preparation of Xenopus egg extracts and demembranated sperm nuclei. We detail how these extracts can be used to study the key transitions of the eukaryotic cell cycle and describe conditions under which these transitions can be manipulated by addition of drugs that either retard or advance passage. In addition, we describe in detail essential techniques that provide a practical starting point for investigating the function of proteins involved in the operation of the eukaryotic cell cycle.

Background Minimal important difference (MID) is a concept used inconsistently and arbitrarily in tendinopathy research. Our aim was to determine the MIDs for the most commonly used tendinopathy outcome measures using data-driven approaches. Methods Recently published systematic reviews of randomised controlled trials (RCTs) on tendinopathy management were identified and used for extraction of eligible studies through a literature search. Each eligible RCT was used to obtain information on MID where this was used and it also contributed data for the calculation of the baseline pooled standard deviation (SD) for each tendinopathy (shoulder, lateral elbow, patellar and Achilles). The rule of “half SD” was used for the computation of MIDs for patient-reported pain (visual analogue scale, VAS 0–10, single-item questionnaire) and function (multi-item questionnaires) and the rule of “one standard error of measurement (SEM)” was additionally used for the multi-item functional outcome measures. Results A total of 119 RCTs were included for the 4 tendinopathies. MID was defined and used by 58 studies (49%) and there were significant inconsistencies amongst studies where the same outcome measure was used as MID. From our data-driven methods the following suggested MIDs were obtained: a) Shoulder tendinopathy, pain VAS (combined) 1.3 points, Constant-Murley score 6.9 (half SD) and 7.0 (one SEM) points; b) lateral elbow tendinopathy, pain VAS (combined) 1.0 point, Disabilities of Arm, Shoulder and Hand questionnaire 8.9 (half SD) and 4.1 (one SEM) points; c) Patellar tendinopathy, pain VAS (combined) 1.2 points, Victorian Institute of Sport Assessment – Patella (VISA-P) 7.3 (half SD) and 6.6 points (one SEM); d) Achilles tendinopathy, pain VAS (combined) 1.1 points, VISA-Achilles (VISA-A) 8.2 (half SD) and 7.8 points (one SEM). The rules of half SD and one SEM produced very similar MIDs except for DASH due to its very high internal consistency. MIDs were also calculated for different pain settings for each tendinopathy. Conclusions Our computed MIDs can be used in tendinopathy research to increase consistency. Clearly defined MIDs should be used with consistency in tendinopathy management studies in the future.

Cells face several challenges to completing genome duplication. One challenge is the irreversible stalling of converging replication forks (double fork stalls). Cell types that cannot delay mitotic entry must also ensure that no replication origins are too far apart (the random gap problem). We show how these challenges can be met in early Xenopus embryos by the very abundant licensing of replication origins: one MCM2-7 double hexamer every ~250 bp. Licensing does not change nucleosome spacing, consistent with MCM2-7 being assembled onto inter-nucleosomal linker DNA. We show that later embryonic development can occur successfully with a per-cell cycle failure rate of <0.2% in early embryos. The high density of licensed origins in the early embryo reduces cell cycle failures from random gaps and from double fork stalls to levels compatible with subsequent development, suggesting that Xenopus early embryonic cells can ensure complete genome duplication without requiring unconventional replication mechanisms.