“Interdisciplinary science” and “breaking down silos” have been buzz phrases for many years. It is encouraging to see that indeed more and more biologists have either teamed up with or adopted approaches from other disciplines to advance scientific progress (Wang et al. 2021). The study of genome biology is no exception. Mechanical forces and structural constraints can have profound effects on nuclear organization, genome integrity, and ultimately cell function. To understand the biological impact of such physical constraints requires not only a physicist’s perspective but also their knowledge and tools. This special issue will highlight the range of mechanical forces that act on and affect the nucleus, from DNA secondary structures and three-dimensional chromatin organization to nuclear compartmentalization via phase separation and the physical deformation of entire nuclei. Together, this collection of papers provides a cross-disciplinary view of the cellular response to DNA damage, DNA replication, and nuclear integrity, shedding new light on our understanding of genome maintenance in normal physiology and in disease.

Our collection starts with a high-resolution view of the chromatin nanostructure that underlies all DNA transactions and dictates overall nuclear organization. Through single-molecule FRET analyses of core histones and their post-translational modifications, original work by Hinde and colleagues illustrates the importance of studying the epigenetic landscape as a function of space within intact nuclear architecture (Liang et al. 2024). The authors find that, while gene-activating or repressive histone marks do overall distinguish open from compact chromatin, there is significant spatial heterogeneity at the level of single chromatin fibers that should be considered when studying chromatin structure in living cells. Finally, this study opens the door for combinatorial analyses of chromatin sub-populations such as bivalent chromatin domains.

Altmeyer and colleagues then take us from nucleosome to nucleus with a review that explores the intersection of genome integrity and mechanobiology (Spegg and Altmeyer 2023). Following a brief discussion of cell cycle regulation, DNA replication, and cellular responses to DNA replication stress, the authors explore the intricate connections between these processes and the biophysical forces that drive DNA repair factor assembly. Using replication protein A (RPA) as an example, the review highlights how repair factors form biophysical condensates, how such condensates can be modulated by DNA damage-induced post-translational modifications, and how this level of control can directly impact DNA repair processes such as the homology-directed Alternative Lengthening of Telomeres (ALT). The review then explores the involvement of nuclear actin filaments and myosin in DNA repair and during replication stress. The authors speculate that actinomyosin networks could serve as molecular highways that direct DNA lesions to optimal repair environments. Intriguingly, lesion mobility appears to extend to DNA repair condensates, emphasizing how distinct biophysical forces can act in concert to ensure accurate genome maintenance. The authors end with a discussion that highlights how the viscoelastic properties that generate and respond to mechanical forces, such as nuclear deformation during cell migration, may directly impact DNA repair efficiency and genome stability. The authors emphasize the need for further research to fully understand the complex interplay between these components and suggest that insights from soft matter physics, polymer mechanics, and material science are likely to contribute significantly to a deeper understanding of genome function in health and disease.

Expanding on the interplay between physical constraints and DNA replication, articles by the Remus and Lopes labs discuss the impact of DNA and RNA secondary structures and higher-order chromatin organization, respectively (Kumar and Remus 2023; González-Acosta and Lopes 2023). Remus and colleagues highlight both known and less appreciated sources of nucleic acid-based replication obstacles, including RNA methylation, RNA:DNA hybrids, topological stress, and G-quadruplex (G4) DNA. The authors emphasize the intricate interrelations between these obstacles as they shift their focus to R loops—three-stranded RNA:DNA structures thought to have both physiological and pathological roles in genome maintenance. A number of DNA and/or RNA-based perturbations are discussed that affect the replication-stalling potential of R loops, either by modulating R loop stability or their intrinsic asymmetry. The authors then dissect the physical nature of DNA polymerase collisions with R loops, which, depending on whether the RNA:DNA hybrid forms on the leading or lagging strand, occur either co-directionally or head-on and translate into distinct biological consequences. A collection of recent findings is discussed that outlines why head-on collisions are generally more disruptive, and how both yeast and human cells may have adapted to this problem through a bias towards the co-orientation of transcription and replication. Inspired by all this progress, the authors stress that future studies need to involve R loops with defined structural attributes to fully comprehend the variety of outcomes of R loop-replisome collisions.

Moving up from nanoscale molecular obstacles, Lopes and colleagues discuss how higher-order nuclear constraints such as chromatin state, genome organization, and nuclear dynamics help orchestrate both replication initiation and replication fork progression. After briefly summarizing the different techniques of genome-wide replication fork mapping, they provide an intriguing and still rather speculative overview of the roles of DNA topology and mobility as organizers of replication zones, and perhaps even distinct replication stress responses. Replication fork reversal is then discussed as a versatile strategy to overcome replication fork stalling and placed in the context of nuclear organization as well as its relation to alternative means of fork recovery, such as repriming and translesion synthesis. Affecting all these studies, the authors raise an important note of caution regarding experimental conditions that induce complete fork arrest. Mild replication stress, the authors argue, is more likely to reflect the effects of certain chemotherapeutic treatment and needs to be a focus of future research efforts. With these limitations in mind, the authors describe an impressive collection of recent work, connecting the replication stress response to various structural elements, from cohesins and topologically associated domains (TADs) to the nuclear lamina, nuclear actin, and replication fork mobility. It is clear from this review that, while physical constraints are a central challenge to replication fork progression, physical forces may be equally important to resolve replication stress. “How” these forces work together remains a topic for future investigations.

Biological consequences of physical constraints are not limited to cell-intrinsic sources but extend to environmental factors, most notably confinement by the micro-environment. For example, work from the Discher lab and others showed that, when applied to cancer cells, physical stress can generate DNA damage and modulate repair factor assembly (Dos Santos and Toseland 2021; Xia et al. 2018). Underlining potential clinical impact, both confinement and tumor cell stiffness relate closely to cancer progression and metastasis of solid tumors (Wei et al. 2017). In new original work, Discher and colleagues now speculate that differences in the biophysical constraints acting on liquid and solid tumors conceivably contribute to differences in genome instability (Wang et al. 2023). Liquid tumors exhibit far fewer chromosome gains and losses when compared to solid tumors. Starting from this intriguing fact, the authors aim to provide a deeper understanding of what limits liquid cancer aneuploidy. Given recent pan-cancer analyses that connect p53 inactivation with aneuploidy, they focus on a p53-deficient leukemia cell line to study Copy Number Variation (CNV) evolution using single-cell DNA sequencing. Their data suggest that factors other than p53 exert stronger pressures against aneuploidy in liquid cancers, and identifying such CNV suppressors could be useful across liquid and solid tumor types. However, they also demonstrate that p53 contributes to sensitivity of liquid cancers to the mechano-environment, which could bias the death of specific genotypes and drive p53-dependent selection of new genotypes, which might in turn play an important role in liquid cancer genetics.

From the nanoscale exploration of chromatin structure to the broader examination of higher-order nuclear constraints, this special issue sheds light on the multifaceted impact of mechanical forces on DNA replication, repair, and nuclear integrity. It also underscores that our understanding of the interplay between mechanobiology and genome maintenance is only starting to unfold. We anticipate that continued efforts to break down disciplinary barriers in scientific inquiry will yield notable advancements in the years ahead.