Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Holton, Nathaniel W; Andrews, Joel; Gassman, Natalie R.

doi:10.3791/56265

Cited by 26 publications

(25 citation statements)

References 47 publications

(72 reference statements)

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“…As XRCC1 has no known enzymatic activity, we assessed DNA repair activity by examining the recruitment and retention of XRCC1 at sites of DNA damage induced by laser microirradiation. Single-strand breaks were induced in a subnuclear region by microirradiation with a 355 nm laser, and we assessed XRCC1 recruitment and retention to the damage sites at various time points after inducing damage [28, 29].…”

Section: Resultsmentioning

confidence: 99%

Defective base excision repair in the response to DNA damaging agents in triple negative breast cancer

Lee

Piett

Andrews

et al. 2019

Preprint

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21 22 23DNA repair defects have been increasingly focused on as therapeutic targets. In hormone 24 positive breast cancer, XRCC1-deficient tumors have been identified and proposed as 25 targets for combination therapies that damage DNA and inhibit DNA repair pathways. 26XRCC1 is a scaffold protein that functions in base excision repair (BER) by mediating 27 essential interactions between DNA glycosylases, AP endonuclease, poly(ADP-ribose) 28 polymerase 1, DNA polymerase β (POL β), and DNA ligases. Loss of XRCC1 confers 29 BER defects and hypersensitivity to DNA damaging agents. BER defects have not been 30 evaluated in triple negative breast cancer (TNBC), for which new therapeutic targets and 31 therapies are needed. To evaluate the potential of XRCC1 as an indicator of BER defects 32 in TNBC, we examined XRCC1 expression and localization in the TCGA database and in 33 TNBC cell lines. High XRCC1 expression was observed for TNBC tumors in the TCGA 34 database and expression of XRCC1 varied between TNBC cell lines. We also observed 35 changes in XRCC1 subcellular localization in TNBCs that alter the ability to repair base 36 lesions and single-strand breaks. Subcellular localization changes were also observed for 37 POL β that did not correlate with XRCC1 localization. Basal levels of DNA damage were 38 also measured in the TNBC cell lines, and damage levels correlated with observed 39 changes in XRCC1 expression, localization, and repair functions. The results confirmed 40 that XRCC1 expression changes may indicate DNA repair capacity changes but 41 emphasize that basal DNA damage levels along with expression and localization are 42 better indicators of DNA repair defects. Given the observed over-expression of XRCC1 in 43 TNBC preclinical models and the TCGA database, XRCC1 expression levels should be 44 considered when evaluating treatment responses of TNBC preclinical model cells. 45 3 Keywords: triple negative breast cancer, base excision repair, XRCC1, PARP1, DNA 46 repair, DNA damage, nuclear localization 47 48 Defects in DNA damage response and repair are driving factors in carcinogenesis 49and key determinants in the response to chemotherapy. Breast cancers may display 50 defects in DNA repair such as mutations in key DNA damage response and repair 51 proteins such as breast cancer-susceptibility (BRCA1/2) and tumor suppressor protein 52 p53 (TP53), and altered expression levels of DNA repair proteins thymine-DNA 53 glycosylase (TDG) and poly(ADP-ribose) polymerase 1 (PARP1) [1-3]. Therapeutic 54 outcomes may be improved by exploiting DNA repair defects present in cancer cells but 55 absent in normal cells, as in the use of PARP-inhibitors (PARPi) in cancers that have 56 BRCA1/2 deficiencies. However, characterization of DNA repair pathways often is lacking 57 in preclinical models and cell lines. Examining DNA repair defects in preclinical models 58 and patients is essential for evaluating the efficacy of therapeutic agents. 59 In breast cancer, DNA repair defects often extend beyond homologous 60 re...

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Section: Resultsmentioning

confidence: 99%

Defective base excision repair in the response to DNA damaging agents in triple negative breast cancer

Lee

Piett

Andrews

et al. 2019

Preprint

Self Cite

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“…Indeed, the quantity and type of DNA lesions generated by micro-irradiation will depend on the laser wavelength and dose as well as on the pre-sensitization methods. Users need to keep in mind that even though DSBs are generated by the conditions described here, 405 nm laser micro-irradiation of photosensitized cells will also generate a mixture of other DNA lesions, including cyclopyrimidine dimers (CPDs), SSBs, and oxidized bases, that will vary between microscope systems and settings 23,24,25 . Thus, a careful monitoring of the lesions at micro-irradiated sites needs to be performed to obtain a comprehensive picture of the damage generated and the response elicited.…”

Section: Discussionmentioning

confidence: 99%

“…Whenever new conditions are being tested, it is convenient to use fluorescently-labeled well-established DNA repair proteins to test doses, wavelengths, and pre-sensitizer combinations, and to ensure that the lesion of interest is efficiently generated. For example, PARP1 and XRCC1 can function as markers for SSB and base-excision repair, XPA is a good indicator of nucleotide excision repair, and 53BP1 will be recruited to DSBs and function in non-homologous end-joining (references 23,25,27,28 ). Antibodies that recognize modifications associated with specific types of DNA damage can also be used to quantify the levels of DNA adducts or breaks.…”

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confidence: 99%

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

Gaudreau-Lapierre

Garneau

Djerir

et al. 2018

JoVE

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The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (γ-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

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“…Objectives with a high numerical aperture should be employed to achieve diffraction-limited focusing and fine micromanipulation. A starting point to plan the application of microirradiation techniques can be found here: ( 148 , 149 ).…”

Section: Laser Microirradiationmentioning

confidence: 99%

Multimodal Light Microscopy Approaches to Reveal Structural and Functional Properties of Promyelocytic Leukemia Nuclear Bodies

et al. 2018

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The promyelocytic leukemia (pml) gene product PML is a tumor suppressor localized mainly in the nucleus of mammalian cells. In the cell nucleus, PML seeds the formation of macromolecular multiprotein complexes, known as PML nuclear bodies (PML NBs). While PML NBs have been implicated in many cellular functions including cell cycle regulation, survival and apoptosis their role as signaling hubs along major genome maintenance pathways emerged more clearly. However, despite extensive research over the past decades, the precise biochemical function of PML in these pathways is still elusive. It remains a big challenge to unify all the different previously suggested cellular functions of PML NBs into one mechanistic model. With the advent of genetically encoded fluorescent proteins it became possible to trace protein function in living specimens. In parallel, a variety of fluorescence fluctuation microscopy (FFM) approaches have been developed which allow precise determination of the biophysical and interaction properties of cellular factors at the single molecule level in living cells. In this report, we summarize the current knowledge on PML nuclear bodies and describe several fluorescence imaging, manipulation, FFM, and super-resolution techniques suitable to analyze PML body assembly and function. These include fluorescence redistribution after photobleaching, fluorescence resonance energy transfer, fluorescence correlation spectroscopy, raster image correlation spectroscopy, ultraviolet laser microbeam-induced DNA damage, erythrocyte-mediated force application, and super-resolution microscopy approaches. Since most if not all of the microscopic equipment to perform these techniques may be available in an institutional or nearby facility, we hope to encourage more researches to exploit sophisticated imaging tools for their research in cancer biology.

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Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Cited by 26 publications

References 47 publications

Defective base excision repair in the response to DNA damaging agents in triple negative breast cancer

Defective base excision repair in the response to DNA damaging agents in triple negative breast cancer

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

Multimodal Light Microscopy Approaches to Reveal Structural and Functional Properties of Promyelocytic Leukemia Nuclear Bodies

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