Summary Error-free repair of DNA double-strand breaks (DSB) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway1. In the absence of BRCA1-mediated HR, administration of PARP inhibitors induces synthetic lethality of tumor cells of patients with breast or ovarian cancers2,3. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration4. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases5. In particular, little is known about BRCA1-independent restoration of HR. Here, we show that loss of REV7 (also known as MAD2L2) re-establishes CtIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and appears to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance6. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining (NHEJ) during immunoglobulin class switch recombination. Our results reveal an unexpected critical function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.

Many genetic processes depend on proteins interacting with specific sequences on DNA. Despite the large excess of nonspecific DNA in the cell, proteins can locate their targets rapidly. After initial nonspecific binding, they are believed to find the target site by 1D diffusion (''sliding'') interspersed by 3D dissociation/reassociation, a process usually referred to as facilitated diffusion. The 3D events combine short intrasegmental ''hops'' along the DNA contour, intersegmental ''jumps'' between nearby DNA segments, and longer volume ''excursions.'' The impact of DNA conformation on the search pathway is, however, still unknown. Here, we show direct evidence that DNA coiling influences the specific association rate of EcoRV restriction enzymes. Using optical tweezers together with a fast buffer exchange system, we obtained association times of EcoRV on single DNA molecules as a function of DNA extension, separating intersegmental jumping from other search pathways. Depending on salt concentration, targeting rates almost double when the DNA conformation is changed from fully extended to a coiled configuration. Quantitative analysis by an extended facilitated diffusion model reveals that only a fraction of enzymes are ready to bind to DNA. Generalizing our results to the crowded environment of the cell we predict a major impact of intersegmental jumps on target localization speed on DNA.A n essential feature in biological processes on DNA is the ability of proteins to quickly locate specific DNA sequences in a vast surplus of nonspecific DNA (1, 2). A protein's search for the target site is thought to be accelerated by facilitated diffusion along nonspecific DNA (3-6). Recent work has yielded considerable insight in the possible search strategies of sitespecific proteins (7-15). Assisted by DNA looping some proteins can, for instance, intermittently bind to two DNA segments simultaneously. This way they can directly move from one to another chemically remote segment (16). This intersegmental transfer accelerates target finding on DNA because it assumes a constantly changing random configuration (11). Here, we demonstrate and quantify a similar mechanism, intersegmental jumping, for proteins with only one DNA-binding site.Little experimental work on facilitated diffusion is available. To date, most studies have been investigating DNA cleavage by restriction enzymes in bulk assays, measuring association times as a function of DNA length, or monitoring processivity on DNA constructs with two sites (7,(17)(18)(19)(20)(21). Although in these biochemical assays valuable information can be obtained, association rates of proteins to specific sites are difficult to measure, and the underlying kinetics of the target search mechanism are often obscured. Furthermore, it remains experimentally challenging to distinguish 1D and 3D search pathways. In previous singlemolecule assays only pure 1D protein search has been addressed (21-24). Here, we present single-molecule measurements of DNA cleavage by EcoRV on individual plasm...

We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes.

When DNA-binding proteins search for their specific binding site on a DNA molecule they alternate between linear 1-dimensional diffusion along the DNA molecule, mediated by nonspecific binding, and 3-dimensional volume excursion events between successive dissociation from and rebinding to DNA. If the DNA molecule is kept in a straight configuration, for instance, by optical tweezers, these 3-dimensional excursions may be divided into long volume excursions and short hops along the DNA. These short hops correspond to immediate rebindings after dissociation such that a rebinding event to the DNA occurs at a site that is close to the site of the preceding dissociation. When the DNA molecule is allowed to coil up, immediate rebinding may also lead to so-called intersegmental jumps, i.e., immediate rebindings to a DNA segment that is far away from the unbinding site when measured in the chemical distance along the DNA, but close by in the embedding 3-dimensional space. This effect is made possible by DNA looping. The significance of intersegmental jumps was recently demonstrated in a single DNA optical tweezers setup. Here we present a theoretical approach in which we explicitly take the effect of DNA coiling into account. By including the spatial correlations of the short hops we demonstrate how the facilitated diffusion model can be extended to account for intersegmental jumping at varying DNA densities. It is also shown that our approach provides a quantitative interpretation of the experimentally measured enhancement of the target location by DNA-binding proteins.gene regulation | search processes | random processes | intersegmental jumps | single-molecule biophysics A t any given instant of time a biological cell only uses part of its genes for production of other molecules (RNA, proteins). During the development of the cell, or the organism the cell belongs to, different genes may be turned on. A classical example is the lactose metabolism of bacteria: in absence of lactose the Lac repressor is specifically bound at the lacZ gene and prevents unnecessary production of the lactose enzyme used to digest the milk sugar; when only lactose and no glucose is present the lacZ gene is activated and lactose-digesting enzymes are produced. Equally famed is the λ switch in Escherichia coli bacteria that are infected by bacteriophage λ. There, the λ switch decides between the dormant lysogenic state or the state of lysis that leads to the production of new phages and ultimate death of the E.coli host cell. Molecularly, the regulation of a gene relies on the presence of certain DNA-binding proteins, so-called transcription factors, that bind to a specific binding site close to the starting sequence of the related gene. The transcription factor then promotes or inhibits binding of RNA polymerase and thus regulates the transcription of this gene. This strategy allows eukaryotic and prokaryotic cells as well as viruses to respond to different internal or external signals (1, 2).The binding of the protein to its specific bindin...