Guidelines for DNA recombination and repair studies: Mechanistic assays of DNA repair processes

Klein, Hannah L.; Ang, Kenny Kean‐Hooi; Arkin, Michelle R.; Beckwitt, Emily C.; Chang, Yi Hsuan; Fan, Jun; Kwon, Youngho; Morten, Michael J.; Mukherjee, Shravanti; Pambos, Oliver J.; Sayyed, Hafez El; Thrall, Elizabeth S.; Vieira-da-Rocha, João Pedro; Wang, Quan; Wang, Shuang; Yeh, Hsin-Yi; Biteen, Julie S.; Chi, Peter; Heyer, Wolf Dietrich; Kapanidis, Achillefs N.; Loparo, Joseph J.; Strick, Térence; Sung, Patrick; Houten, Bennett Van; Niu, Hengyao; Rothenberg, Eli

doi:10.15698/mic2019.01.665

Cited by 11 publications

(6 citation statements)

References 184 publications

(277 reference statements)

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“…Rad55-Rad57 foci were induced by 40 Gy using an X-ray irradiator (Faxitron,. Interfoci fluorescence redistribution after photobleaching (iFRAP) was conducted as described previously (Klein et al, 2019). In brief, exponentially growing cells were exposed to 40 Gy, which induces an average of 2 DSBs per cell (Lisby et al, 2001), and cultured shaking for another 3 hours at 25˚C prior to iFRAP.…”

Section: Live Cell Imaging Of Rad55-rad57 Focimentioning

confidence: 99%

The Rad51 paralog complex Rad55-Rad57 acts as a molecular chaperone during homologous recombination

et al. 2021

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Homologous recombination (HR) is essential for the maintenance of genome integrity. Rad51 paralogs fulfill a conserved, but undefined role in HR, and their mutations are associated with increased cancer risk in humans. Here, we use single-molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55-Rad57 promotes the assembly of Rad51 recombinase filaments through transient interactions, providing evidence that it acts as a classical molecular chaperone. Srs2 is an ATP-dependent anti-recombinase that downregulates HR by actively dismantling Rad51 filaments. Contrary to the current model, we find that Rad55-Rad57 does not physically block the movement of Srs2. Instead, Rad55-Rad57 promotes rapid reassembly of Rad51 filaments after their disruption by Srs2. Our findings support a model in which Rad51 is in flux between free and ssDNA-bound states, the rate of which is dynamically controlled though the opposing actions of Rad55-Rad57 and Srs2.

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Section: Live Cell Imaging Of Rad55-rad57 Focimentioning

confidence: 99%

The Rad51 paralog complex Rad55-Rad57 acts as a molecular chaperone during homologous recombination

et al. 2021

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“…Among these DNA repair pathways, homologous recombination (HR) is one of the key pathways for the repair of DSBs [7]. A variety of DDR proteins are involved in the regulation of HR, resulting in cancer drug resistance [8].…”

Section: Introductionmentioning

confidence: 99%

MiR-7-5p-mediated downregulation of PARP1 impacts DNA homologous recombination repair and resistance to doxorubicin in small cell lung cancer

et al. 2019

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Background Chemo-resistance is one of the major challenges in the therapy of small cell lung cancer (SCLC). Multiple mechanisms are thought to be involved in chemo-resistance during SCLC treatment, but unfortunately, these mechanisms have not been well elucidated. Herein, we investigated the role of miRNA in the resistance of SCLC cells to doxorubicin (Dox). Methods MiRNA microarray analysis revealed that several miRNAs, including miR-7-5p, were specifically decreased in Dox-resistant SCLC cells (H69AR) compared to parental cells (H69). The expression level of miR-7-5p was confirmed by qRT-PCR in Dox-resistant cells (H69AR and H446AR cells) and their parental cells. Bioinformatic analysis indicated that poly ADP-ribose polymerase 1 (PARP1) is a direct target of miR-7-5p. The binding sites of miR-7-5p in the PARP1 3′ UTR were verified by luciferase reporter and Western blot assays. To investigate the role of miR-7-5p in the chemo-resistance of SCLC cells to doxorubicin, mimic or inhibitor of miR-7-5p was transfected into SCLC cells, and the effect of miR-7-5p on homologous recombination (HR) repair was analyzed by HR reporter assays. Furthermore, the expression of HR repair factors (Rad51 and BRCA1) induced by doxorubicin was detected by Western blot and immunofluorescent staining in H446AR cells transfected with miR-7-5p mimic. Results The expression level of miR-7-5p was remarkably reduced ( -fold) in Dox-resistant SCLC cells (H69AR and H446AR cells) compared with that in parental cells (H69 and H446 cells). Poly ADP-ribose polymerase 1 (PARP1) is a direct target of miR-7-5p, and PARP1 expression was downregulated by miR-7-5p. MiR-7-5p impeded Dox-induced HR repair by inhibiting the expression of HR repair factors (Rad51 and BRCA1) that resulted in resensitizing SCLC cells to doxorubicin. Conclusions Our findings provide evidence that miR-7-5p targets PARP1 to exert its suppressive effects on HR repair, indicating that the alteration of the expression of miR-7-5p may be a promising strategy for overcoming chemo-resistance in SCLC therapy. Electronic supplementary material The online version of this article (10.1186/s12885-019-5798-7) contains supplementary material, which is available to authorized users.

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“…31 Combination of such supercoiled DNA with single-molecule magnetic trap assay allows one to detect the torsional changes of DNA imposed by RNAP or other proteins through detecting the changes in DNA extension. It gives a spatial resolution of ∼1 bp at a temporal resolution of 2−3 s. 35,41 As ∼100 molecules can be monitored in real time in one field of view under the microscope, this assay allows a high-throughput data collection fashion for detecting the dynamic interactions between RNAP and DNA. A 2.2 kb linear DNA is used in our experimentation, which contains a centrally located T5N25 promoter followed by a 97 bp transcription unit and an E. coli his terminator (Figure 2a).…”

Section: Transcription Characterized Via Single-molecule Magnetic Tra...mentioning

confidence: 99%

Correlative Escherichia coli Transcription Rate and Bubble Conformation Remodeled by NusA and NusG

et al. 2023

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Transcription is highly regulated by a variety of transcription factors, among which NusA and NusG act contradictorily in Escherichia coli (E. coli) that NusA stabilizes a paused RNA polymerase (RNAP) and NusG suppresses it. The mechanism of the NusA and NusG regulations on RNAP transcription has been addressed, but their effect on the conformational changes of the transcription bubble correlated with transcription kinetics remains elusive. By using single-molecule magnetic trap, we identify a reduction in the transcription rate of ∼40% events by NusA. Although the rest ∼60% of transcription events exhibit unaffected transcription rates, a NusA-enhanced standard deviation of the transcription rate is observed. NusA remodeling also increases the extent of DNA unwinding in the transcription bubble by 1–2 base pairs, which can be reduced by NusG. The NusG remodeling is more significant on the RNAP molecules with reduced transcription rates rather than those without. Our results provide a quantitative view on the mechanisms of transcriptional regulation by NusA and NusG factors.

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Guidelines for DNA recombination and repair studies: Mechanistic assays of DNA repair processes

Cited by 11 publications

References 184 publications

The Rad51 paralog complex Rad55-Rad57 acts as a molecular chaperone during homologous recombination

The Rad51 paralog complex Rad55-Rad57 acts as a molecular chaperone during homologous recombination

MiR-7-5p-mediated downregulation of PARP1 impacts DNA homologous recombination repair and resistance to doxorubicin in small cell lung cancer

Correlative Escherichia coli Transcription Rate and Bubble Conformation Remodeled by NusA and NusG

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