CRISPR-Cas immunity, DNA repair and genome stability

Co-opting of CRISPR-Cas ‘Interference’ reactions for editing the genomes of eukaryotic and prokaryotic cells has highlighted crucial support roles for DNA repair systems that strive to maintain genome stability. As front-runners in genome editing that targets DNA, the class 2 CRISPR-Cas enzymes Cas9 and Cas12a rely on repair of DNA double-strand breaks (DDSBs) by host DNA repair enzymes, using mechanisms that vary in how well they are understood. Data are emerging about the identities of DNA repair enzymes that support genome editing in human cells. At the same time, it is becoming apparent that CRISPR-Cas systems functioning in their native environment, bacteria or archaea, also need DNA repair enzymes. In this short review, we survey how DNA repair and CRISPR-Cas systems are intertwined. We consider how understanding DNA repair and CRISPR-Cas interference reactions in nature might help improve the efficacy of genome editing procedures that utilise homologous or analogous systems in human and other cells.

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Tim–Tipin dysfunction creates an indispensible reliance on the ATR–Chk1 pathway for continued DNA synthesis

The Journal of Cell Biology ◽

10.1083/jcb.200905006 ◽

2009 ◽

Vol 187 (1) ◽

pp. 15-23 ◽

Cited By ~ 52

Author(s):

Kevin D. Smith ◽

Michael A. Fu ◽

Eric J. Brown

Keyword(s):

Dna Synthesis ◽

Genome Stability ◽

Double Strand Breaks ◽

Dna Unwinding ◽

Replication Forks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Nucleotide Incorporation ◽

Pathway Activation ◽

Maintain Genome Stability

The Tim (Timeless)–Tipin complex has been proposed to maintain genome stability by facilitating ATR-mediated Chk1 activation. However, as a replisome component, Tim–Tipin has also been suggested to couple DNA unwinding to synthesis, an activity expected to suppress single-stranded DNA (ssDNA) accumulation and limit ATR–Chk1 pathway engagement. We now demonstrate that Tim–Tipin depletion is sufficient to increase ssDNA accumulation at replication forks and stimulate ATR activity during otherwise unperturbed DNA replication. Notably, suppression of the ATR–Chk1 pathway in Tim–Tipin-deficient cells completely abrogates nucleotide incorporation in S phase, indicating that the ATR-dependent response to Tim–Tipin depletion is indispensible for continued DNA synthesis. Replication failure in ATR/Tim-deficient cells is strongly associated with synergistic increases in H2AX phosphorylation and DNA double-strand breaks, suggesting that ATR pathway activation preserves fork stability in instances of Tim–Tipin dysfunction. Together, these experiments indicate that the Tim–Tipin complex stabilizes replication forks both by preventing the accumulation of ssDNA upstream of ATR–Chk1 function and by facilitating phosphorylation of Chk1 by ATR.

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DNA repair goes hip-hop: SMARCA and CHD chromatin remodellers join the break dance

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2016.0285 ◽

2017 ◽

Vol 372 (1731) ◽

pp. 20160285 ◽

Cited By ~ 27

Author(s):

Magdalena B. Rother ◽

Haico van Attikum

Keyword(s):

Dna Repair ◽

Posttranslational Modifications ◽

Chromatin Structure ◽

Hip Hop ◽

Genome Stability ◽

Genome Instability ◽

Current Knowledge ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks

Proper signalling and repair of DNA double-strand breaks (DSB) is critical to prevent genome instability and diseases such as cancer. The packaging of DNA into chromatin, however, has evolved as a mere obstacle to these DSB responses. Posttranslational modifications and ATP-dependent chromatin remodelling help to overcome this barrier by modulating nucleosome structures and allow signalling and repair machineries access to DSBs in chromatin. Here we recap our current knowledge on how ATP-dependent SMARCA- and CHD-type chromatin remodellers alter chromatin structure during the signalling and repair of DSBs and discuss how their dysfunction impacts genome stability and human disease. This article is part of the themed issue ‘Chromatin modifiers and remodellers in DNA repair and signalling’.

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Homologous recombination repair intermediates promote efficient de novo telomere addition at DNA double-strand breaks

Nucleic Acids Research ◽

10.1093/nar/gkz1109 ◽

2019 ◽

Vol 48 (3) ◽

pp. 1271-1284 ◽

Cited By ~ 4

Author(s):

Anoushka Davé ◽

Chen-Chun Pai ◽

Samuel C Durley ◽

Lydia Hulme ◽

Sovan Sarkar ◽

...

Keyword(s):

Genome Stability ◽

De Novo ◽

Developmental Process ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Recombination Repair ◽

Chromosome Arm ◽

Maintain Genome Stability ◽

Genetic Context

Abstract The healing of broken chromosomes by de novo telomere addition, while a normal developmental process in some organisms, has the potential to cause extensive loss of heterozygosity, genetic disease, or cell death. However, it is unclear how de novo telomere addition (dnTA) is regulated at DNA double-strand breaks (DSBs). Here, using a non-essential minichromosome in fission yeast, we identify roles for the HR factors Rqh1 helicase, in concert with Rad55, in suppressing dnTA at or near a DSB. We find the frequency of dnTA in rqh1Δ rad55Δ cells is reduced following loss of Exo1, Swi5 or Rad51. Strikingly, in the absence of the distal homologous chromosome arm dnTA is further increased, with nearly half of the breaks being healed in rqh1Δ rad55Δ or rqh1Δ exo1Δ cells. These findings provide new insights into the genetic context of highly efficient dnTA within HR intermediates, and how such events are normally suppressed to maintain genome stability.

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Dot1L-dependent H3K79 methylation facilitates histone variant H2A.Z exchange at DNA double strand breaks and is required for high fidelity, homology-directed DNA repair

10.1101/544981 ◽

2019 ◽

Author(s):

Nehemiah S. Alvarez ◽

Pavla Brachova ◽

Timothy A. Fields ◽

Patrick E. Fields

Keyword(s):

Dna Repair ◽

Genome Stability ◽

Repair Process ◽

Histone Variant ◽

Double Strand Breaks ◽

Open Chromatin ◽

High Fidelity ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Non Homologous End Joining

AbstractIn eukaryotic cells, the homology-directed repair (HDR) and non-homologous end joining (NHEJ) pathways are required for the repair of DNA double strand breaks (DSB). The high-fidelity HDR pathway is particularly important for maintenance of genomic stability. In mammals, histone post-translational modifications and histone variant exchange into nucleosomes at sites of DSB generate an open chromatin state necessary for repair to take place. However, the specific contributions of histone modifications to histone variant exchange at DSB sites and the influence of these changes on the DNA repair process and genome stability are incompletely understood. Here we show that Dot1L-catalyzed methylation of H3 histone on lysine 79 (H3K79) is required for efficient HDR of DSB. In cells with DNA DSB either lacking Dot1L or expressing a methylation-dead Dot1L, there is altered kinetics of DNA repair factor recruitment, markedly decreased H2A.Z incorporation at DSB sites, and a specific and profound reduction in HDR, which results in significant genomic instability. These findings demonstrate a new role for Dot1L, identifying it as a critical regulator of the DNA repair process and a steward of genomic integrity.

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Recruitment of lysine demethylase 2A to DNA double strand breaks and its interaction with 53BP1 ensures genome stability

Oncotarget ◽

10.18632/oncotarget.24636 ◽

2018 ◽

Vol 9 (22) ◽

pp. 15915-15930 ◽

Cited By ~ 5

Author(s):

Murilo T.D. Bueno ◽

Marta Baldascini ◽

Stéphane Richard ◽

Noel F. Lowndes

Keyword(s):

Genome Stability ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Lysine Demethylase

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End-resection at DNA double-strand breaks in the three domains of life

Biochemical Society Transactions ◽

10.1042/bst20120307 ◽

2013 ◽

Vol 41 (1) ◽

pp. 314-320 ◽

Cited By ~ 30

Author(s):

John K. Blackwood ◽

Neil J. Rzechorzek ◽

Sian M. Bray ◽

Joseph D. Maman ◽

Luca Pellegrini ◽

...

Keyword(s):

Dna Repair ◽

Homologous Recombination ◽

Strand Break ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Domains Of Life ◽

Strand Invasion ◽

End Resection

During DNA repair by HR (homologous recombination), the ends of a DNA DSB (double-strand break) must be resected to generate single-stranded tails, which are required for strand invasion and exchange with homologous chromosomes. This 5′–3′ end-resection of the DNA duplex is an essential process, conserved across all three domains of life: the bacteria, eukaryota and archaea. In the present review, we examine the numerous and redundant helicase and nuclease systems that function as the enzymatic analogues for this crucial process in the three major phylogenetic divisions.

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i-BLESS is an ultra-sensitive method for detection of DNA double-strand breaks

Communications Biology ◽

10.1038/s42003-018-0165-9 ◽

2018 ◽

Vol 1 (1) ◽

Cited By ~ 22

Author(s):

Anna Biernacka ◽

Yingjie Zhu ◽

Magdalena Skrzypczak ◽

Romain Forey ◽

Benjamin Pardo ◽

...

Keyword(s):

Cell Fate ◽

Genome Stability ◽

Strand Break ◽

Double Strand Breaks ◽

Universal Method ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Agarose Beads ◽

Genome Wide ◽

Dna Double Strand Break

AbstractMaintenance of genome stability is a key issue for cell fate that could be compromised by chromosome deletions and translocations caused by DNA double-strand breaks (DSBs). Thus development of precise and sensitive tools for DSBs labeling is of great importance for understanding mechanisms of DSB formation, their sensing and repair. Until now there has been no high resolution and specific DSB detection technique that would be applicable to any cells regardless of their size. Here, we present i-BLESS, a universal method for direct genome-wide DNA double-strand break labeling in cells immobilized in agarose beads. i-BLESS has three key advantages: it is the only unbiased method applicable to yeast, achieves a sensitivity of one break at a given position in 100,000 cells, and eliminates background noise while still allowing for fixation of samples. The method allows detection of ultra-rare breaks such as those forming spontaneously at G-quadruplexes.

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

Nucleic Acids Research ◽

10.1093/nar/gkaa713 ◽

2020 ◽

Vol 48 (17) ◽

pp. 9449-9461

Author(s):

Lea Milling Korsholm ◽

Zita Gál ◽

Blanca Nieto ◽

Oliver Quevedo ◽

Stavroula Boukoura ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Damage Response ◽

Ribosomal Dna ◽

Repetitive Sequences ◽

Dna Lesions ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Damage Response

Abstract 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.

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