The Use of Laser Microirradiation to Investigate the Roles of Cohesins in DNA Repair

Kong, Xiangduo; Ball, Alexander R.; Yokomori, Kyoko

doi:10.1007/978-1-4939-6545-8_14

Cited by 4 publications

(3 citation statements)

References 59 publications

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“…Cohesin proteins have major roles in a number of fundamental molecular processes, including sister chromatid cohesion, transcriptional control and DNA dsb repair. In recombinational repair of DNA dsbs, there is genetic and biochemical evidence from multiple species that cohesin accumulates around the site of the dsb and stabilizes it for subsequent enzymatic activity by homologous recombination enzymes (25)(26)(27)(28). Cohesin is a multiprotein complex with four core components:…”

Section: Discussionmentioning

confidence: 99%

A Roberts Syndrome Individual With Differential Genotoxin Sensitivity and a DNA Damage Response Defect

McKay

Craig

Kalitsis

et al. 2019

International Journal of Radiation Oncology*Biology*Physics

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Syndrome individual with differential genotoxin sensitivity and a DNA damage response defect, SUMMARY Purpose: Roberts Syndrome (RBS) is a rare recessively-transmitted developmental disorder characterized by growth retardation, craniofacial abnormalities and truncation of limbs. All affected individuals to date have mutations in the ESCO2 (Establishment of cohesion 2) gene, a key regulator of the cohesin complex, which is involved in sister chromatid cohesion and DNA double-strand break (dsb) repair.Here we characterize DNA damage responses (DDRs) for the first time in a RBSaffected family. Methods and Materials: Lymphoblastoid cell lines (LCLs) were established from an RBS family, including the proband, and parents carrying ESCO2 mutations. Various DDR assays were performed on these cells, including clonogenic, chromosome break and apoptosis assays, checkpoint activation indicators and measures of DNA breakage and repair. Results: Cells derived from the RBS-affected individual showed sensitivity to ionizing radiation (IR) and Mitomycin C (MMC) -induced DNA damage. In this ESCO2 compound heterozygote, other DNA damage responses were also defective, including enhanced IR-induced clastogenicity and apoptosis, increased DNA dsb induction and a reduced capacity for repairing IR -induced DNA dsbs as measured by γ-H2AX foci and the comet assay.Conclusions: in addition to its developmental features, RBS can be, like ataxia telangiectasia, considered a DNA damage response-defective syndrome, which contributes to its cellular, molecular and clinical phenotype.

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Section: Discussionmentioning

confidence: 99%

A Roberts Syndrome Individual With Differential Genotoxin Sensitivity and a DNA Damage Response Defect

McKay

Craig

Kalitsis

et al. 2019

International Journal of Radiation Oncology*Biology*Physics

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“…One of the most successful applications of LS-OT has been the trapping and/or damaging of (mitotic) chromosomes. This kind of studies will keep providing critical information on mitosis kinetics, regulation and consequences of chromosomal dysfunctionality ( Kong et al, 2017 ; Milas et al, 2018 ; Odell et al, 2019 ). For example, a very recent publication reports on a very fine level of LS disruption of particular mitotic machinery elements in order to better understand mitotic chromosome migration ( Forer and Berns, 2020 ).…”

Section: Perspectivesmentioning

confidence: 99%

Genetic Material Manipulation and Modification by Optical Trapping and Nanosurgery-A Perspective

Blázquez‐Castro

Fernández‐Piqueras

Santos

2020

Front. Bioeng. Biotechnol.

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Light can be employed as a tool to alter and manipulate matter in many ways. An example has been the implementation of optical trapping, the so called optical tweezers, in which light can hold and move small objects with 3D control. Of interest for the Life Sciences and Biotechnology is the fact that biological objects in the size range from tens of nanometers to hundreds of microns can be precisely manipulated through this technology. In particular, it has been shown possible to optically trap and move genetic material (DNA and chromatin) using optical tweezers. Also, these biological entities can be severed, rearranged and reconstructed by the combined use of laser scissors and optical tweezers. In this review, the background, current state and future possibilities of optical tweezers and laser scissors to manipulate, rearrange and alter genetic material (DNA, chromatin and chromosomes) will be presented. Sources of undesirable effects by the optical procedure and measures to avoid them will be discussed. In addition, first tentative approaches at cellular-level genetic and organelle surgery, in which genetic material or DNA-carrying organelles are extracted out or introduced into cells, will be presented.

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“…To assess the localization of Rep68/78 to sites of cellular DNA damage in another way, we determined the localization of ectopically expressed Rep by immunofluorescence within seconds following the induction of DNA damage by laser microirradiation (Kong et al, 2017;Mistrik et al, 2016). As shown in Fig.…”

Section: Dna Damagementioning

confidence: 99%

Interactions of parvoviruses with the cellular DNA damage response and CTCF

Boftsi¹

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Nuclear DNA viruses must be able to simultaneously access cellular factors that aid their life cycle while evading inhibitory factors in order to sustain their expression and replication. Establishment of nuclear sites where particular DNA viruses are able to initiate their replication has likely evolved over time to optimize these needs. Parvoviruses are small non-enveloped icosahedral viruses that are important pathogens in many animal species including humans. They contain a linear single-stranded DNA genome of ~5 kb with Inverted Terminal Repeats (ITRs) at each end that serve as origins of replication. Minute Virus of Mice (MVM), the prototype virus of the genus Protoparvovirus, is an autonomously replicating mouse parvovirus that is lytic in murine cells and human transformed cells. Unlike most parvoviruses, Adeno-associated virus (AAV) in the genus Dependoparvovirus cannot support their own replication and require the coinfection of a helper virus, such as Adenovirus or Herpesvirus, in order to propagate. Parvoviruses establish replication factories in the nucleus, where ongoing replication and expression takes place. Due to their limited genetic capacity, parvoviruses depend heavily on cellular functions and must interact with host factors (and helper virus factors in case of AAV) either positively or negatively to productively replicate. Parvovirus infection induces a cellular DNA damage response (DDR) in cells, either in response to viral DNA or viral proteins expressed in the host nucleus during infection. Using a novel adaptation of high-throughput circular chromosome conformation capture assay (4C) for use in trans (which we have termed V3C-seq), we have mapped the primary localization sites of the replicating MVM genome with cellular sites of DNA damage, replete with replication and repair factors. As replication proceeds, MVM induces and then spreads to additional sites of DNA damage forming large interaction domains termed Virus Association Domains (VADs). Furthermore, we have shown that the essential viral non-structural protein NS1 possess the intrinsic ability to localize to cellular DNA damage sites and could localize to those sites both the MVM genome and a heterologous DNA molecule engineered to contain NS1 binding sites. These results suggested that NS1 may function as a bridging molecule, helping the MVM genome localize to sites of DNA damage to facilitate ongoing virus replication. Similar V3C-seq assays to map AAV genome localization show that both replicating and non-replicating AAV2 genomes in the absence of helper virus colocalize with cellular sites of DNA damage. The AAV non-structural protein Rep68/78, when ectopically expressed in the absence of viral infection or during AAV2 infection in the absence of helper proteins also localizes to cellular sites of DNA damage. Strikingly however, recombinant AAV gene therapy vector genomes derived from AAV do not colocalize with AAV and Rep at cellular DDR sites, suggesting the Inverted Terminal Repeat origins of replication shared between them are insufficient to localize the genome to cellular sites of DNA damage. Our findings suggest that AAV Rep78 protein plays another critical role in the AAV life cycle by aiding in the establishment of viral replication centers in proximity to cellular sites of DNA damage. Our V3C-seq analysis on MVM also revealed that the VADs overlap significantly with previously identified topologically-associated domains (TADs). The boundaries of TADs are enriched for binding sites of CCCTC-binding factor (CTCF), a highly conserver 11-zinc-finger DNA-binding protein which play a key role in genome organization. In silico inspection of the MVM genome revealed the presence of two potential CTCF binding sites and Chromatin Immunoprecipitation (ChIP) assays showed that CTCF associates specifically with the viral genome. In our attempt to determine the role of the host factor CTCF during MVM infection, we found that the multifunctional host factor CTCF is required for proper engagement of the spliceosome at the MVM small intron and for the first steps of processing of P4 promoter-generated pre-mRNAs.

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The Use of Laser Microirradiation to Investigate the Roles of Cohesins in DNA Repair

Cited by 4 publications

References 59 publications

A Roberts Syndrome Individual With Differential Genotoxin Sensitivity and a DNA Damage Response Defect

A Roberts Syndrome Individual With Differential Genotoxin Sensitivity and a DNA Damage Response Defect

Genetic Material Manipulation and Modification by Optical Trapping and Nanosurgery-A Perspective

Interactions of parvoviruses with the cellular DNA damage response and CTCF

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