Studying DNA Double-Strand Break Repair: An Ever-Growing Toolbox

Vítor, Alexandra C; Huertas, Pablo; Legube, Gaëlle; Almeida, Sérgio Fernandes de

doi:10.3389/fmolb.2020.00024

Cited by 124 publications

(96 citation statements)

References 145 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…HR is slower and requires a homologous DNA sequence to act as a template for accurate repair ( 70 ). Although NHEJ is less accurate than HR and can lead to loss of genetic information, it can occur throughout the cell cycle, whereas HR is generally restricted to S and G2 phases, when a sister chromatid is available ( 65 , 69 , 71 , 72 ). One of the first steps towards HR is initiation of resection by the MRN-complex, consisting of MRE11, RAD50 and NBS1 and CtIP ( 73 ).…”

Section: Introductionmentioning

confidence: 99%

“…At the site of damage MDC1 interacts with the MRN complex ( 78 ), ensuring increased MRN accumulation at the break site and thereby creating a positive feedback loop amplifying ATM recruitment ( 77 ). Multiple modifications of the surrounding chromatin lead to the formation of ionizing radiation induced foci that facilitate accumulation of proteins involved in processing and repair of the DSB ( 65 ).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Recent advances in the nucleolar responses to DNA double-strand breaks

Korsholm

Gál

Nieto

et al. 2020

Nucleic Acids Research

View full text Add to dashboard Cite

DNA damage poses a serious threat to human health and cells therefore continuously monitor and repair DNA lesions across the genome. Ribosomal DNA is a genomic domain that represents a particular challenge due to repetitive sequences, high transcriptional activity and its localization in the nucleolus, where the accessibility of DNA repair factors is limited. Recent discoveries have significantly extended our understanding of how cells respond to DNA double-strand breaks (DSBs) in the nucleolus, and new kinases and multiple down-stream targets have been identified. Restructuring of the nucleolus can occur as a consequence of DSBs and new data point to an active regulation of this process, challenging previous views. Furthermore, new insights into coordination of cell cycle phases and ribosomal DNA repair argue against existing concepts. In addition, the importance of nucleolar-DNA damage response (n-DDR) mechanisms for maintenance of genome stability and the potential of such factors as anti-cancer targets is becoming apparent. This review will provide a detailed discussion of recent findings and their implications for our understanding of the n-DDR. The n-DDR shares features with the DNA damage response (DDR) elsewhere in the genome but is also emerging as an independent response unique to ribosomal DNA and the nucleolus.

show abstract

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Recent advances in the nucleolar responses to DNA double-strand breaks

Korsholm

Gál

Nieto

et al. 2020

Nucleic Acids Research

View full text Add to dashboard Cite

show abstract

“…A second advantage of the dCas9-KR system is that, because ROS have a short (nanoseconds) half-life in cells (42,43), termination of light exposure quickly removes the source of DNA damage, providing the ability to monitor repair in the absence of ongoing damage. Other approaches to generating DSBs with targeted nucleases, including Cas9 (27), have limitations due to the time taken for induction or expression of the enzyme, which can be 1-5 hr (44). Further, because these approaches usually lead to constitutive expression of the nuclease, cells engage in multiple cutrepair cycles, until errors accumulate that eliminate the target site (29,45).…”

Section: Discussionmentioning

confidence: 99%

“…Targeted DSBs have been used to identify histone modifications and patterns of histone exchange following DNA damage (4,5,28), while Cas9/gRNA has been used widely to monitor repair mechanism pathways and the fidelity of DNA repair (27,29). These approaches have provided invaluable insights into how the DNA repair machinery works in concert with the chromatin.…”

Section: Introductionmentioning

confidence: 99%

Site-specific targeting of a light activated dCas9-KIllerRed fusion protein generates transient, localized regions of oxidative DNA damage

House

Layer

Price

2020

Preprint

View full text Add to dashboard Cite

DNA repair requires reorganization of the local chromatin structure to facilitate access to and repair of the DNA. Studying DNA double-strand break (DSB) repair in specific chromatin domains has been aided by the use of sequence-specific endonucleases to generate targeted breaks. Here, we describe a new approach that combines KillerRed, a photosensitizer that generates reactive oxygen species (ROS) when exposed to light, and the genome-targeting properties of the CRISPR/Cas9 system. Fusing KillerRed to catalytically inactive Cas9 (dCas9) generates dCas9-KR, which can then be targeted to any desired genomic region with an appropriate guide RNA. Activation of dCas9-KR with green light generates a local increase in reactive oxygen species, resulting in “clustered” oxidative damage, including both DNA breaks and base damage. Activation of dCas9-KR rapidly (within minutes) increases both gH2AX and recruitment of the KU70/80 complex. Importantly, this damage is repaired within 10 minutes of termination of light exposure, indicating that the DNA damage generated by dCas9-KR is both rapid and transient. Further, repair is carried out exclusively through NHEJ, with no detectable contribution from HR-based mechanisms. Surprisingly, sequencing of repaired DNA damage regions did not reveal any increase in either mutations or INDELs in the targeted region, implying that NHEJ has high fidelity under the conditions of low level, limited damage. The dCas9-KR approach for creating targeted damage has significant advantages over the use of endonucleases, since the duration and intensity of DNA damage can be controlled in “real time” by controlling light exposure. In addition, unlike endonucleases that carry out multiple cut-repair cycles, dCas9-KR produces a single burst of damage, more closely resembling the type of damage experienced during acute exposure to reactive oxygen species or environmental toxins. dCas9-KR is a promising system to induce DNA damage and measure site-specific repair kinetics at clustered DNA lesions.

show abstract

“…For example, depending on the DNA repair pathway elicited, the resection of DSBs exposes ssDNA that fall under the purview of ATR [11,73]. Classical and alternative DNA repair pathways, such as homologous recombination, nonhomologous end joining (NHEJ), or break-induced replication repair, may be employed to allow fork restart [74][75][76]. When repair is not possible, the mechanisms of damage tolerance may be activated to restart the fork, such as the lesion bypass (where the replisome "skips" the lesion and resumes replication downstream), template switch (a homologous recombination (HR)-mediated response that involves fork regression and the use of the newly synthesized DNA strand as a template) and translesion synthesis (TLS), in which specialized polymerases capable of bypassing the lesion are recruited [77][78][79].…”

Section: Overview Of Dna Replication Stress: How Cells Prevent and Rementioning

confidence: 99%

Protective Mechanisms Against DNA Replication Stress in the Nervous System

Charlier

Martins

2020

Genes

View full text Add to dashboard Cite

The precise replication of DNA and the successful segregation of chromosomes are essential for the faithful transmission of genetic information during the cell cycle. Alterations in the dynamics of genome replication, also referred to as DNA replication stress, may lead to DNA damage and, consequently, mutations and chromosomal rearrangements. Extensive research has revealed that DNA replication stress drives genome instability during tumorigenesis. Over decades, genetic studies of inherited syndromes have established a connection between the mutations in genes required for proper DNA repair/DNA damage responses and neurological diseases. It is becoming clear that both the prevention and the responses to replication stress are particularly important for nervous system development and function. The accurate regulation of cell proliferation is key for the expansion of progenitor pools during central nervous system (CNS) development, adult neurogenesis, and regeneration. Moreover, DNA replication stress in glial cells regulates CNS tumorigenesis and plays a role in neurodegenerative diseases such as ataxia telangiectasia (A-T). Here, we review how replication stress generation and replication stress response (RSR) contribute to the CNS development, homeostasis, and disease. Both cell-autonomous mechanisms, as well as the evidence of RSR-mediated alterations of the cellular microenvironment in the nervous system, were discussed.

show abstract

Studying DNA Double-Strand Break Repair: An Ever-Growing Toolbox

Cited by 124 publications

References 145 publications

Recent advances in the nucleolar responses to DNA double-strand breaks

Recent advances in the nucleolar responses to DNA double-strand breaks

Site-specific targeting of a light activated dCas9-KIllerRed fusion protein generates transient, localized regions of oxidative DNA damage

Protective Mechanisms Against DNA Replication Stress in the Nervous System

Contact Info

Product

Resources

About