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RAD51 protects against nonconservative DNA double-strand break repair through a nonenzymatic function.

ABSTRACT: Selection of the appropriate DNA double-strand break (DSB) repair pathway is decisive for genetic stability. It is proposed to act according to two steps: 1-canonical nonhomologous end-joining (C-NHEJ) versus resection that generates single-stranded DNA (ssDNA) stretches; 2-on ssDNA, gene conversion (GC) versus nonconservative single-strand annealing (SSA) or alternative end-joining (A-EJ). Here, we addressed the mechanisms by which RAD51 regulates this second step, preventing nonconservative repair in human cells. Silencing RAD51 or BRCA2 stimulated both SSA and A-EJ, but not C-NHEJ, validating the two-step model. Three different RAD51 dominant-negative forms (DN-RAD51s) repressed GC and stimulated SSA/A-EJ. However, a fourth DN-RAD51 repressed SSA/A-EJ, although it efficiently represses GC. In living cells, the three DN-RAD51s that stimulate SSA/A-EJ failed to load efficiently onto damaged chromatin and inhibited the binding of endogenous RAD51, while the fourth DN-RAD51, which inhibits SSA/A-EJ, efficiently loads on damaged chromatin. Therefore, the binding of RAD51 to DNA, rather than its ability to promote GC, is required for SSA/A-EJ inhibition by RAD51. We showed that RAD51 did not limit resection of endonuclease-induced DSBs, but prevented spontaneous and RAD52-induced annealing of complementary ssDNA in vitro. Therefore, RAD51 controls the selection of the DSB repair pathway, protecting genome integrity from nonconservative DSB repair through ssDNA occupancy, independently of the promotion of CG.

SUBMITTER: So A

PROVIDER: S-EPMC8934640 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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RAD51 protects against nonconservative DNA double-strand break repair through a nonenzymatic function.

So Ayeong A Dardillac Elodie E Muhammad Ali A Chailleux Catherine C Sesma-Sanz Laura L Ragu Sandrine S Le Cam Eric E Canitrot Yvan Y Masson Jean Yves JY Dupaigne Pauline P Lopez Bernard S BS Guirouilh-Barbat Josée J

Nucleic acids research 20220301 5

Selection of the appropriate DNA double-strand break (DSB) repair pathway is decisive for genetic stability. It is proposed to act according to two steps: 1-canonical nonhomologous end-joining (C-NHEJ) versus resection that generates single-stranded DNA (ssDNA) stretches; 2-on ssDNA, gene conversion (GC) versus nonconservative single-strand annealing (SSA) or alternative end-joining (A-EJ). Here, we addressed the mechanisms by which RAD51 regulates this second step, preventing nonconservative re ...[more]

PMID: 35137208

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Project description:Expression of RAD51, a crucial player in homologous recombination (HR) and DNA double-strand break (DSB) repair, is dysregulated in human tumors, and can contribute to genomic instability and tumor progression. To further understand RAD51 regulation we functionally characterized a long non-coding (lnc) RNA, dubbed TODRA (Transcribed in the Opposite Direction of RAD51), transcribed 69bp upstream to RAD51, in the opposite direction. We demonstrate that TODRA is an expressed transcript and that the RAD51 promoter region is bidirectional, supporting TODRA expression (7-fold higher than RAD51 in this assay, p = 0.003). TODRA overexpression in HeLa cells induced expression of TPIP, a member of the TPTE family which includes PTEN. Similar to PTEN, we found that TPIP co-activates E2F1 induction of RAD51. Analysis of E2F1's effect on the bidirectional promoter showed that E2F1 binding to the same site that promotes RAD51 expression, results in downregulation of TODRA. Moreover, TODRA overexpression induces HR in a RAD51-dependent DSB repair assay, and increases formation of DNA damage-induced RAD51-positive foci. Importantly, gene expression in breast tumors supports our finding that E2F1 oppositely regulates RAD51 and TODRA: increased RAD51 expression, which is associated with an aggressive tumor phenotype (e.g. negative correlation with positive ER (r = -0.22, p = 0.02) and positive PR status (r = -0.27, p<0.001); positive correlation with ki67 status (r = 0.36, p = 0.005) and HER2 amplification (r = 0.41, p = 0.001)), correlates as expected with lower TODRA and higher E2F1 expression. However, although E2F1 induction resulted in TPIP downregulation in cell lines, we find that TPIP expression in tumors is not reduced despite higher E2F1 expression, perhaps contributing to increased RAD51 expression. Our results identify TPIP as a novel E2F1 co-activator, suggest a similar role for other TPTEs, and indicate that the TODRA lncRNA affects RAD51 dysregulation and RAD51-dependent DSB repair in malignancy. Importantly, gene expression in breast tumors supports our finding that E2F1 oppositely regulates RAD51 and TODRA: increased RAD51 expression, which is associated with an aggressive tumor phenotype (e.g. negative correlation with positive ER (r = -0.22, p = 0.02) and positive PR status (r = -0.27, p<0.001); positive correlation with ki67 status (r = 0.36, p = 0.005) and HER2 amplification (r = 0.41, p = 0.001)), correlates as expected with lower TODRA and higher E2F1 expression. However, although E2F1 induction resulted in TPIP downregulation in cell lines, we find that TPIP expression in tumors is not reduced despite higher E2F1 expression, perhaps contributing to increased RAD51 expression. Our results identify TPIP as a novel E2F1 co-activator, suggest a similar role for other TPTEs, and indicate that the TODRA lncRNA affects RAD51 dysregulation and RAD51-dependent DSB repair in malignancy.

Dataset Information

RAD51 protects against nonconservative DNA double-strand break repair through a nonenzymatic function.

Publications

RAD51 protects against nonconservative DNA double-strand break repair through a nonenzymatic function.

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