The study of protein recruitment to laser-induced DNA lesions can be distorted by photoconversion of the DNA binding dye Hoechst

Hurst, Verena; Gasser, Susan M.

doi:10.12688/f1000research.17865.2

Cited by 4 publications

(4 citation statements)

References 24 publications

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“…Combined with the observation that the p53 mutant, R248Q, did not accumulate at the damage site (Fig. 1C, Movie S3), we validated that our system was free of photoconversion artifacts, as discussed elsewhere (16).…”

Section: Resultssupporting

confidence: 80%

Rapid recruitment of p53 to DNA damage sites directs DNA repair choice and integrity

Wang

Hariharan

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Significance Our work focuses on the critical longstanding question of the nontranscriptional role of p53 in tumor suppression. We demonstrate here that poly(ADP-ribose) polymerase (PARP)–dependent modification of p53 enables rapid recruitment of p53 to damage sites, where it in turn directs early repair pathway selection. Specifically, p53-mediated recruitment of 53BP1 at early time points promotes nonhomologous end joining over the more error-prone microhomology end-joining. Similarly, p53 directs nucleotide excision repair by mediating DDB1 recruitment. This property of p53 also correlates with tumor suppression in vivo. Our study provides mechanistic insight into how certain transcriptionally deficient p53 mutants may retain tumor-suppressive functions through regulating the DNA damage response.

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Section: Resultssupporting

confidence: 80%

Rapid recruitment of p53 to DNA damage sites directs DNA repair choice and integrity

Wang

Hariharan

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…Images of the nuclear stain should be captured last, after images are collected for other fluorophores. Although 405 nm laser excitation can induce photoconversion as previously reported [4], it required longer exposure time and high laser power (Figure 3). When using confocal microscopy, one can avoid photoconversion by navigating to fields not exposed to UV light before capturing images.…”

Section: Discussionmentioning

confidence: 60%

“…Both green and red fluorescent products have been detected after UV exposure of DAPI and Hoechst [3]. Photoconversion of Hoechst also has been shown to occur with 405 nm laser excitation, a potential complication for studies using Hoechst dyes to photosensitize living cells to UV-induced DNA damage [4].…”

Section: Introductionmentioning

confidence: 99%

Tips for Avoiding Artifacts from UV Photoconversion of DAPI

Roberts¹

2019

Micros. Today

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Exposure of the nuclear counterstain DAPI to just a few seconds of UV excitation light during fluorescence imaging can photoconvert the dye to stable green-and red-emitting forms, resulting in imaging artifacts in multi-color staining experiments. This article reviews the published literature on photoconversion and compares the effect of different mounting media on photoconversion of DAPI in stained tissue sections. Once photoconversion is recognized as a potential source of non-specific fluorescence, careful imaging practices or reagent selection can be used to avoid it.

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“…From a technical point of view, we verified that the increase of green fluorescent signal on the laser track was not due to Hoechst photoconversion ( Figure 2G ), a phenomenon in which UV irradiation can induce a chemical change in the Hoechst molecule that leads to emission of green and red wavelengths ( Hurst and Gasser, 2019 ).…”

Section: Anticipated Resultsmentioning

confidence: 85%

Imaging the Response to DNA Damage in Heterochromatin Domains

Chansard

Pobega

Caron

et al. 2022

Front. Cell Dev. Biol.

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The eukaryotic genome is assembled in a nucleoprotein complex called chromatin, whose organization markedly influences the repair of DNA lesions. For instance, compact chromatin states, broadly categorized as heterochromatin, present a challenging environment for DNA damage repair. Through transcriptional silencing, heterochromatin also plays a vital role in the maintenance of genomic integrity and cellular homeostasis. It is thus of critical importance to decipher whether and how heterochromatin affects the DNA damage response (DDR) to understand how this chromatin state is preserved after DNA damage. Here, we present two laser micro-irradiation-based methods for imaging the DDR in heterochromatin domains in mammalian cells. These methods allow DNA damage targeting to specific subnuclear compartments, direct visualization of the DDR and image-based quantification of the repair response. We apply them to study DNA double-strand break repair pathways in facultative heterochromatin and the repair of UV photoproducts in constitutive heterochromatin. We discuss the advantages and limitations of these methods compared to other targeted approaches for DNA damage induction.

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The study of protein recruitment to laser-induced DNA lesions can be distorted by photoconversion of the DNA binding dye Hoechst

Cited by 4 publications

References 24 publications

Rapid recruitment of p53 to DNA damage sites directs DNA repair choice and integrity

Rapid recruitment of p53 to DNA damage sites directs DNA repair choice and integrity

Tips for Avoiding Artifacts from UV Photoconversion of DAPI

Imaging the Response to DNA Damage in Heterochromatin Domains

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