DNA Strand Exchange to Monitor Human RAD51-Mediated Strand Invasion and Pairing

Lahiri, Sudipta; Jensen, Ryan B.

doi:10.1007/978-1-0716-0644-5_8

Cited by 3 publications

(4 citation statements)

References 25 publications

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“…The gel was then dried onto DE81 paper and exposed to a PhosphorImager screen overnight. The percentage of DNA strand exchange product was calculated as labeled product divided by total labeled input DNA in each lane (for further details see Jensen et al, 2010; Lahiri and Jensen, 2021).…”

Section: Methodsmentioning

confidence: 99%

“…2XMBP-BRC2, BRC7, S1221P-BRC2, T1980I-BRC7 or BRCA2 wild-type, S1221P, T1346I and T1980I (see table below for amino acid sequences) were purified as described for the purification full-length BRCA2 protein (Jensen et al, 2010;Lahiri and Jensen, 2021). The phCMV1 constructs with human BRCA2 cDNA and 2XMBP tag was transiently transfected using CaPO4 precipitation into HEK293T cells, which were harvested 30 hours posttransfection.…”

Section: Protein Purificationmentioning

confidence: 99%

“…The copyright holder for this preprint this version posted September 25, 2021. ; https://doi.org/10.1101/2021.09.24.461749 doi: bioRxiv preprint purification full-length BRCA2 protein (Jensen et al, 2010;Lahiri and Jensen, 2021). The phCMV1 constructs with human BRCA2 cDNA and 2XMBP tag was transiently transfected using CaPO4 precipitation into HEK293T cells, which were harvested 30 hours posttransfection.…”

Section: Protein Purificationmentioning

confidence: 99%

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BRCA2 BRC missense variants disrupt RAD51-dependent DNA repair

Jiménez-Sáinz

Mathew

Garbarino

et al. 2021

Preprint

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BRCA2 is a tumor suppressor gene that maintains genome stability by mediating the high fidelity repair of DNA double-strand breaks (DSBs) through homology-directed repair (HDR). Pathogenic mutations in BRCA2 predispose to breast, ovarian, pancreatic, prostate, and other cancers. Mutations in BRCA2 leading to severe protein truncation predict pathogenicity, however, missense mutations with unknown functional consequences, designated Variants of Uncertain Significance (VUS), comprise 60% of BRCA2 sequence changes deposited in clinical databases. Classifying BRCA2 VUS correctly is critical for relaying clinically actionable information to patients concerning future cancer risk or current treatment options. In this study, we identified and biochemically characterized three BRCA2 VUS located in BRC repeats to determine the impact on canonical HDR functions. Two of the germline variants, S1221P and T1980I, map to conserved residues in BRC2 and BRC7, disrupt RAD51 binding, and are diminished in their ability to stabilize RAD51-ssDNA complexes. We provide supporting cellular evidence that S1221P and T1980I are significantly compromised in their response to chemotherapeutics and ionizing radiation. The third variant, T1346I, lies within the spacer region between BRC2 and BRC3 but remains fully functional. We conclude that T1346I has a neutral impact on BRCA2 function, while S1221P and T1980I are hypomorphic alleles that disrupt the ability of BRCA2 to fully engage and stabilize RAD51 nucleoprotein filaments.

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Section: Methodsmentioning

confidence: 99%

Section: Protein Purificationmentioning

confidence: 99%

Section: Protein Purificationmentioning

confidence: 99%

See 1 more Smart Citation

BRCA2 BRC missense variants disrupt RAD51-dependent DNA repair

Jiménez-Sáinz

Mathew

Garbarino

et al. 2021

Preprint

Self Cite

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“…In mammalian cells, DNA DSBs repair is carried out by two canonical mechanisms: fast but incorrect, non-homologous end joining (NHEJ) and slow, but relatively correct, homologous recombination (HR) and several alternative pathways [25]. The mechanistic steps of HR-dependent repair involve DNA end resection, strand invasion, homology search, and pairing, followed by new DNA synthesis [26]. NHEJ contributes to repair of about 80% of DNA DSBs [27].…”

Section: Introductionmentioning

confidence: 99%

The CD44high Subpopulation of Multifraction Irradiation-Surviving NSCLC Cells Exhibits Partial EMT-Program Activation and DNA Damage Response Depending on Their p53 Status

Pustovalova

Alhaddad

Blokhina

et al. 2021

IJMS

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Ionizing radiation (IR) is used for patients diagnosed with unresectable non-small cell lung cancer (NSCLC). However, radiotherapy remains largely palliative due to the survival of specific cell subpopulations. In the present study, the sublines of NSCLC cells, A549IR (p53wt) and H1299IR (p53null) survived multifraction X-ray radiation exposure (MFR) at a total dose of 60 Gy were investigated three weeks after the MFR course. We compared radiosensitivity (colony formation), expression of epithelial-mesenchymal transition (EMT) markers, migration activity, autophagy, and HR-dependent DNA double-strand break (DSB) repair in the bulk and entire CD44high/CD166high CSC-like populations of both parental and MFR survived NSCLC cells. We demonstrated that the p53 status affected: the pattern of expression of N-cadherin, E-cadherin, Vimentin, witnessing the appearance of EMT-like phenotype of MFR-surviving sublines; 1D confined migratory behavior (wound healing); the capability of an irradiated cell to continue to divide and form a colony of NSCLC cells before and after MFR; influencing the CD44/CD166 expression level in MFR-surviving NSCLC cells after additional single irradiation. Our data further emphasize the impact of p53 status on the decay of γH2AX foci and the associated efficacy of the DSB repair in NSCLC cells survived after MFR. We revealed that Rad51 protein might play a principal role in MFR-surviving of p53 null NSCLC cells promoting DNA DSB repair by homologous recombination (HR) pathway. The proportion of Rad51 + cells elevated in CD44high/CD166high population in MFR-surviving p53wt and p53null sublines and their parental cells. The p53wt ensures DNA-PK-mediated DSB repair for both parental and MFR-surviving cells irrespectively of a subsequent additional single irradiation. Whereas in the absence of p53, a dose-dependent increase of DNA-PK-mediated non-homologous end joining (NHEJ) occurred as an early post-irradiation response is more intensive in the CSC-like population MFR-surviving H1299IR, compared to their parental H1299 cells. Our study strictly observed a significantly higher content of LC3 + cells in the CD44high/CD166high populations of p53wt MFR-surviving cells, which enriched the CSC-like cells in contrast to their p53null counterparts. The additional 2 Gy and 5 Gy X-ray exposure leads to the dose-dependent increase in the proportion of LC3 + cells in CD44high/CD166high population of both parental p53wt and p53null, but not MFR-surviving NSCLC sublines. Our data indicated that autophagy is not necessarily associated with CSC-like cells’ radiosensitivity, emphasizing that careful assessment of other milestone processes (such as senescence and autophagy-p53-Zeb1 axis) of primary radiation responses may provide new potential targets modulated for therapeutic benefit through radiosensitizing cancer cells while rescuing normal tissue. Our findings also shed light on the intricate crosstalk between autophagy and the p53-related EMT, by which MFR-surviving cells might obtain an invasive phenotype and metastatic potential.

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BRCA2 BRC missense variants disrupt RAD51-dependent DNA repair

Jiménez-Sáinz

Mathew

Moore

et al. 2022

eLife

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Pathogenic mutations in the BRCA2 tumor suppressor gene predispose to breast, ovarian, pancreatic, prostate, and other cancers. BRCA2 maintains genome stability through homology-directed repair (HDR) of DNA double-strand breaks (DSBs) and replication fork protection. Nonsense or frameshift mutations leading to truncation of the BRCA2 protein are typically considered pathogenic, however, missense mutations resulting in single amino acid substitutions can be challenging to functionally interpret. The majority of missense mutations in BRCA2 have been classified as Variants of Uncertain Significance (VUS) with unknown functional consequences. In this study, we identified three BRCA2 VUS located within the BRC repeat region to determine their impact on canonical HDR and fork protection functions. We provide evidence that S1221P and T1980I, which map to conserved residues in the BRC2 and BRC7 repeats, compromise the cellular response to chemotherapeutics and ionizing radiation, and display deficits in fork protection. We further demonstrate biochemically that S1221P and T1980I disrupt RAD51 binding and diminish the ability of BRCA2 to stabilize RAD51-ssDNA complexes. The third variant, T1346I, located within the spacer region between BRC2 and BRC3 repeats, is fully functional. We conclude that T1346I is a benign allele whereas S1221P and T1980I are hypomorphic disrupting the ability of BRCA2 to fully engage and stabilize RAD51 nucleoprotein filaments. Our results underscore the importance of correctly classifying BRCA2 VUS as pathogenic variants can impact both future cancer risk and guide therapy selection during cancer treatment.

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DNA Strand Exchange to Monitor Human RAD51-Mediated Strand Invasion and Pairing

Cited by 3 publications

References 25 publications

BRCA2 BRC missense variants disrupt RAD51-dependent DNA repair

BRCA2 BRC missense variants disrupt RAD51-dependent DNA repair

The CD44high Subpopulation of Multifraction Irradiation-Surviving NSCLC Cells Exhibits Partial EMT-Program Activation and DNA Damage Response Depending on Their p53 Status

BRCA2 BRC missense variants disrupt RAD51-dependent DNA repair

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