Strand-specific ChIP-sequencing reveals nucleosome dynamics at DNA double-strand breaks

DNA double-strand breaks (DSBs) are highly toxic lesions that, if not correctly repaired, can have detrimental consequences on genome integrity and cell survival. During the course of evolution, different mechanisms developed to repair DSBs including nonhomologous end-joining (NHEJ) and homologous recombination (HR). A pivotal step of the cellular repair pathway decision is the processing of DSB ends during DNA end resection, which, through the degradation of 5’-terminated strands, generates a long stretch of single-stranded DNA (ssDNA) that prevents re-ligation by NHEJ factors and guides repair towards HR. At the same time, resection divides the chromatin surrounding a DSB in distinct ssDNA and dsDNA domains. Establishment of these domains is crucial for HR repair as well as for DNA damage signaling and checkpoint activation, but the protein composition and the interactions between these compartments were not fully understood. Specifically, it was unclear whether nucleosomes, the fundamental unit of chromatin, could be found in the ssDNA domain as well. This would have considerable implications for the recruitment of repair factors to DSBs and for the maintenance of the epigenetic information during repair. However, previously used techniques were inadequate to address this question. Here, we combined site-specific induction of DSBs with chromatin immunoprecipitation, followed by strand-specific library preparation and next-generation sequencing to analyze the in vivo DNA binding mode of key DSB repair proteins as well as nucleosomes. In proof-of-principle experiments, strand-specific ChIP-sequencing recapitulated the characteristic binding pattern of RPA and Rad51 to ssDNA at resected DSBs. Using this technique, we were also able to detect Rad51 binding to dsDNA during homology search. The 9-1-1 signaling platform was suggested to bind at the ss-dsDNA junction at resected DSBs. We showed that, in vivo, 9-1-1 associates with the dsDNA compartment and locates at the leading edge of resection. Furthermore, we did not find evidence of the presence of nucleosomes on ssDNA and, therefore, they do not represent a major species at resected DSBs. In contrast, we found that nucleosomes become fully evicted in concomitance with resection and that the chromatin remodelers RSC and SWI/SNF act redundantly to promote such nucleosome eviction. Taken together, our study revealed that nucleosome eviction is intrinsically coupled with resection and that the ssDNA and dsDNA domains generated by resection are characterized by distinct properties

NUCLEOSOMES DYNAMICS AT DNA DOUBLE STRAND BREAKS

Background: DNA double-strand breaks (DSBs) are highly toxic lesions that, if not correctly repaired, can have detrimental consequences on genome integrity and cell survival. Repair of DSBs by homologous recombination (HR) requires the processing of DSB ends during DNA end resection, which, through the degradation of 5’-terminated strands, generates a long stretch of single-stranded DNA (ssDNA). Therefore, resection divides the chromatin surrounding a DSB in distinct ssDNA and dsDNA domains. The molecular composition of these domains is crucial for HR repair as well as for DNA damage signaling and checkpoint activation. However, it was unclear whether nucleosomes, the fundamental unit of chromatin, could be found in the ssDNA domain as well. [...

Unscheduled DNA replication in G1 causes genome instability and damage signatures indicative of replication collisions

DNA replicates once per cell cycle. Interfering with the regulation of DNA replication initiation generates genome instability through over-replication and has been linked to early stages of cancer development. Here, we engineer genetic systems in budding yeast to induce unscheduled replication in a G1- like cell cycle state. Unscheduled G1 replication initiates at canonical S-phase origins. We quantifiy the composition of replisomes in G1- and S-phase and identified firing factors, polymerase α, and histone supply as factors that limit replication outside S-phase. G1 replication per se does not trigger cellular checkpoints. Subsequent replication during S-phase, however, results in overreplication and leads to chromosome breaks and chromosome-wide, strandbiased occurrence of RPA-bound single-stranded DNA, indicating head-to-tail replication collisions as a key mechanism generating genome instability upon G1 replication. Low-level, sporadic induction of G1 replication induces an identical response, indicating findings from synthetic systems are applicable to naturally occurring scenarios of unscheduled replication initiation

Interplay between nucleosomes, nucleases and cell cycle kinases during DNA end resection

DNA double strand breaks (DSBs) can be repaired by two principal cellular mechanisms – recombination-based (such as homologous recombination, HR) and direct ligation-based (such as non-homologous end joining, NHEJ). In mitotically dividing cells HR critically depends on the presence of sister chromatids as repair templates and hence DSB repair pathway choice is highly cell cycle regulated. Regulation occurs primarily at the level of DNA end resection, the first step in the HR reaction. Notably, nucleosomes restrict resection both directly and indirectly by recruiting nucleosome-associated resection inhibitors such as Rad9/53BP1. Nucleosome remodellers – in particular Fun30/SMARCAD1– are recruited to DSBs in order to overcome this inhibition. Our data show how nucleosome remodellers control DNA end resection. Fun30 is under cell cycle control and targeted to DSBs in a cell cycle-regulated manner. Such targeting is efficient long-range resection of DSBs and remarkably, allows to partially bypass the cell cycle regulation of long-range resection, if made constitutive. This suggests that nucleosome remodelling by Fun30 is a critical and conserved mechanism for DSB repair regulation. In order to investigate the fate of nucleosomes during DNA end resection, we established a ChIP-seq-based methodology to measure ssDNA and dsDNA binding of histones in vivo. Our data suggest that nucleosomes are evicted during DNA end resection. A model how nucleosomes may control steps downstream of resection will be discussed. Lastly, we describe a new layer of cell cycle-regulation in the whole process, deregulation of which allows to partially reconstitute DNA end resection and homologous recombination already in the G1 phase of the cell cycle. We will discuss implications for genome editing

Dbf4-dependent kinase (DDK) promotes resection of DNA double strand breaks and repair via homologous recombination

DNA double strand breaks (DSBs) can be repaired via different cellular mechanisms, with two types of repair: direct ligation-based mechanisms or recombination-based mechanisms, like homologous recombination (HR). Ligation-based mechanisms require little processing of the broken ends, while extensive processing via the nucleolytic degradation of the 5’-terminated broken strand (DNA end resection) is crucial to commit cells to recombination-mediated repair. HR makes use of a homologous template, preferentially the sister chromatid, to direct the repair of the DSB and is therefore upregulated in cell cycle phases where the sister chromatid is available. DNA end resection is upregulated in the same cell cycle phases as well and is under strict cell cycle control. Different resection proteins were shown to be activated during the cell cycle by cyclin-dependent kinase (CDK), in both yeast and human. However, CDK-phosphomimetic mutants of these factors does not seem to be able to bypass the cell cycle regulation of DNA end resection. This suggests that additional cell cycle kinases might be important for this regulation. [...

Dbf4-dependent kinase (DDK) promotes resection of DNA double strand breaks and repair via homologous recombination

DNA double strand breaks (DSBs) can be repaired via different cellular mechanisms, with two types of repair: direct ligation-based mechanisms or recombination-based mechanisms, like homologous recombination (HR). Ligation-based mechanisms require little processing of the broken ends, while extensive processing via the nucleolytic degradation of the 5’-terminated broken strand (DNA end resection) is crucial to commit cells to recombination-mediated repair. HR makes use of a homologous template, preferentially the sister chromatid, to direct the repair of the DSB and is therefore upregulated in cell cycle phases where the sister chromatid is available. DNA end resection is upregulated in the same cell cycle phases as well and is under strict cell cycle control. Different resection proteins were shown to be activated during the cell cycle by cyclin-dependent kinase (CDK), in both yeast and human. However, CDK-phosphomimetic mutants of these factors does not seem to be able to bypass the cell cycle regulation of DNA end resection. This suggests that additional cell cycle kinases might be important for this regulation. [...

Unscheduled DNA replication in G1 causes genome instability and damage signatures indicative of replication collisions

DNA replicates once per cell cycle. Interfering with the regulation of DNA replication initiation generates genome instability through over-replication and has been linked to early stages of cancer development. Here, we engineered genetic systems in budding yeast to induce unscheduled replication in the G1-phase of the cell cycle. Unscheduled G1 replication initiated at canonical S-phase origins. We quantified the composition of replisomes in G1- and S-phase and identified firing factors, polymerase α, and histone supply as factors that limit replication outside S-phase. G1 replication per se did not trigger cellular checkpoints. Subsequent replication during S-phase, however, resulted in over-replication and led to chromosome breaks and chromosome-wide, strand-biased occurrence of RPA-bound single-stranded DNA indicating head-to-tail replication fork collisions as key mechanism generating genome instability upon G1 replication. Low-level, sporadic induction of G1 replication induced an identical response, indicating findings from synthetic systems are applicable to naturally occurring scenarios of unscheduled replication initiation

Unscheduled DNA replication in G1 causes genome instability and damage signatures indicative of replication collisions

DNA replicates once per cell cycle. Interfering with the regulation of DNA replication initiation generates genome instability through over-replication and has been linked to early stages of cancer development. Here, we engineered genetic systems in budding yeast to induce unscheduled replication in the G1-phase of the cell cycle. Unscheduled G1 replication initiated at canonical S-phase origins. We quantified the composition of replisomes in G1- and S-phase and identified firing factors, polymerase α, and histone supply as factors that limit replication outside S-phase. G1 replication per se did not trigger cellular checkpoints. Subsequent replication during S-phase, however, resulted in over-replication and led to chromosome breaks and chromosome-wide, strand-biased occurrence of RPA-bound single-stranded DNA indicating head-to-tail replication fork collisions as key mechanism generating genome instability upon G1 replication. Low-level, sporadic induction of G1 replication induced an identical response, indicating findings from synthetic systems are applicable to naturally occurring scenarios of unscheduled replication initiation

Dbf4-dependent kinase promotes cell cycle controlled resection of DNA double-strand breaks and repair by homologous recombination

DNA double-strand breaks (DSBs) can be repaired by several pathways. In eukaryotes, DSB repair pathway choice occurs at the level of DNA end resection and is controlled by the cell cycle. Upon cell cycle-dependent activation, cyclin-dependent kinases (CDKs) phosphorylate resection proteins and thereby stimulate end resection and repair by homologous recombination (HR). However, inability of CDK phospho-mimetic mutants to bypass this cell cycle regulation, suggests that additional cell cycle regulators may be important. Here, we identify Dbf4-dependent kinase (DDK) as a second major cell cycle regulator of DNA end resection. Using inducible genetic and chemical inhibition of DDK in budding yeast and human cells, we show that end resection and HR require activation by DDK. Mechanistically, DDK phosphorylates at least two resection nucleases in budding yeast: the Mre11 activator Sae2, which promotes resection initiation, as well as the Dna2 nuclease, which promotes resection elongation. Notably, synthetic activation of DDK allows limited resection and HR in G1 cells, suggesting that DDK is a key component of DSB repair pathway selection.ISSN:2041-172