Determination of the number of RAD51 molecules in different human cell lines

Foertsch, Franziska; Kache, Tom; Drube, Sebastian; Biskup, Christoph; Nasheuer, Heinz-Peter; Melle, Christian

doi:10.1080/15384101.2019.1691802

Cited by 5 publications

(5 citation statements)

References 31 publications

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“…In CMM patients' cells, the WT-RAD51 protein is reduced (42-67% of WT-RAD51 levels in the intrafamilial controls) without any phenotypic evidence for alterations in its homologous recombination activity: in our CMM cohort, none of the patients with RAD51 pathogenic variants presented fertility problems, Fanconi anemia, or cancer. In line with this observation, the cellular levels of RAD51 are thought to exceed the cell's needs for homologous recombination (35). Our findings strongly support that both truncating and non-truncating RAD51-CMM variants result in a loss of function selectively associated with the CMM phenotype.…”

Section: Discussionsupporting

confidence: 80%

Congenital mirror movements are associated with defective polymerisation of RAD51

Trouillard¹,

Dupaigne²,

Dunoyer³

et al. 2023

J Med Genet

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BackgroundMirror movements are involuntary movements of one hand that mirror intentional movements of the other hand. Congenital mirror movements (CMM) is a rare genetic disorder with autosomal dominant inheritance, in which mirror movements are the main neurological manifestation. CMM is associated with an abnormal decussation of the corticospinal tract, a major motor tract for voluntary movements. RAD51 is known to play a key role in homologous recombination with a critical function in DNA repair. WhileRAD51haploinsufficiency was first proposed to explain CMM, other mechanisms could be involved.MethodsWe performed Sanger sequencing ofRAD51in five newly identified CMM families to identify new pathogenic variants. We further investigated the expression of wild-type and mutant RAD51 in the patients’ lymphoblasts at mRNA and protein levels. We then characterised the functions of RAD51 altered by non-truncating variants using biochemical approaches.ResultsThe level of wild-type RAD51 protein was lower in the cells of all patients with CMM compared with their non-carrier relatives. The reduction was less pronounced in asymptomatic carriers.In vitro, mutant RAD51 proteins showed loss-of-function for polymerisation, DNA binding and strand exchange activity.ConclusionOur study demonstrates thatRAD51haploinsufficiency, including loss-of-function of non-truncating variants, results in CMM. The incomplete penetrance likely results from post-transcriptional compensation. Changes in RAD51 levels and/or polymerisation properties could influence guidance of the corticospinal axons during development. Our findings open up new perspectives to understand the role of RAD51 in neurodevelopment.

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Section: Discussionsupporting

confidence: 80%

Congenital mirror movements are associated with defective polymerisation of RAD51

Trouillard¹,

Dupaigne²,

Dunoyer³

et al. 2023

J Med Genet

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“…HR of DNA repair in eukaryotic cells is mainly conducted by two recombinases, Rad51 and Dmc1. Rad51 is not only responsible for DNA double-strand break repair in mitosis, for example, repairing interrupted replication forks occurring in G2/S phase ( 63 , 64 ) but has also been identified during meiosis to facilitate HR mediated by Dmc1 ( 18 , 65 ). However, the only function of Dmc1 is to mediate HR between parental chromosome for genetic diversity in meiosis I ( 66 ).…”

Section: Discussionmentioning

confidence: 99%

Mechanisms of distinctive mismatch tolerance between Rad51 and Dmc1 in homologous recombination

Zhao

Peng

et al. 2021

Nucleic Acids Research

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Homologous recombination (HR) is a primary DNA double-strand breaks (DSBs) repair mechanism. The recombinases Rad51 and Dmc1 are highly conserved in the RecA family; Rad51 is mainly responsible for DNA repair in somatic cells during mitosis while Dmc1 only works during meiosis in germ cells. This spatiotemporal difference is probably due to their distinctive mismatch tolerance during HR: Rad51 does not permit HR in the presence of mismatches, whereas Dmc1 can tolerate certain mismatches. Here, the cryo-EM structures of Rad51–DNA and Dmc1–DNA complexes revealed that the major conformational differences between these two proteins are located in their Loop2 regions, which contain invading single-stranded DNA (ssDNA) binding residues and double-stranded DNA (dsDNA) complementary strand binding residues, stabilizing ssDNA and dsDNA in presynaptic and postsynaptic complexes, respectively. By combining molecular dynamic simulation and single-molecule FRET assays, we identified that V273 and D274 in the Loop2 region of human RAD51 (hRAD51), corresponding to P274 and G275 of human DMC1 (hDMC1), are the key residues regulating mismatch tolerance during strand exchange in HR. This HR accuracy control mechanism provides mechanistic insights into the specific roles of Rad51 and Dmc1 in DNA double-strand break repair and may shed light on the regulatory mechanism of genetic recombination in mitosis and meiosis.

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“…We also attempted to follow by live imaging the recruitment of another repair factor, RAD51, involved in DSB repair by homologous recombination (HR). However, even when transfecting cells with a plasmid encoding GFP-RAD51 under the control of a weak promoter ( Foertsch et al, 2019 ), the levels of expression were still too high and led to the formation of RAD51 filaments even in undamaged cells, which hampered the study of RAD51 relocalization after laser micro-irradiation. Instead, we assessed the recruitment of two endogenous HR repair factors, phosphorylated RPA (pRPA32 Ser4/8) and RAD51, by immunofluorescence in RPE-1 cells stably expressing macroH2A1.2-GFP ( Figure 2H ).…”

Section: Anticipated Resultsmentioning

confidence: 99%

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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Determination of the number of RAD51 molecules in different human cell lines

Cited by 5 publications

References 31 publications

Congenital mirror movements are associated with defective polymerisation of RAD51

Congenital mirror movements are associated with defective polymerisation of RAD51

Mechanisms of distinctive mismatch tolerance between Rad51 and Dmc1 in homologous recombination

Imaging the Response to DNA Damage in Heterochromatin Domains

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