analysis-nonhomologous-end-joining-homologous-recombination

Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells Using GFP-Based Reporter Systems

DNA double-strand breaks (DSBs) represent the most perilous DNA lesions, capable of inducing substantial genetic information loss and cellular demise. In ...

Qiqihar Medical University

Anterior gradient-2 (AGR2) is a proto-oncogene involved in tumorigenesis and cancer progression. AGR2, predominantly localized in the endoplasmic reticulum (ER), is also a secreted protein detected in the extracellular compartment in multiple cancers. However, the biological functions of intracellular and extracellular AGR2 remain to be elucidated.

Effects of ER-resident and secreted AGR2 on cell proliferation, migration, invasion, and survival in PANC-1 pancreatic cancer cells.

Objective To investigate the interaction between Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) and heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and its biological functions. Methods The interaction between WASH/FAM21, the core member of WASH complex, and hnRNP A1 was identified by mass spectrometry and co-immunoprecipitation. Telomere lengths of shWASH and shScramble cells were measured by Real-time qPCR. RNA-seq was used to generate the transcription profiles of shWASH and shScramble cells. Results Mass spectrometry results indicated that a variety of hnRNP family members, including hnRNP A1, interact with FAM21-d219N. The interaction between endogenous WASH/FAM21 and hnRNP A1 was confirmed by co-immunoprecipitation results. Depletion of WASH did not affect the relative length of telomeres, but it significantly increased the exon skipping and led to the variation of transcriptional expression of hundreds of genes, including some NF-κB target genes. Conclusion WASH in the nucleus interacts with hnRNP A1 to participate in the regulation of alternative splicing and gene transcription.

[WASH regulates alternative splicing and gene transcription by interacting with hnRNP A1].

The Wiskott-Aldrich syndrome protein and SCAR homolog (WASH), an actin nucleation-promoting factor, is present in the nucleus where it regulates gene transcription and maintains nuclear organization. Here, we show that WASH interacts with core non-homologous end-joining (NHEJ) factors including Ku70/Ku80 and DNA-PKcs, and Ku70/Ku80 is involved in the recruitment of WASH to the sites of DNA double-stranded break (DSB). WASH depletion leads to increased cell sensitivity and impaired DNA repair capacity in response to etoposide-induced DSBs and reduces NHEJ efficiency. Mechanistically, we show that loss of WASH inhibits the phosphorylation of DNA-PKcs, H2AX, and KAP1 after DSB induction and reduces chromatin relaxation and the recruitment of several downstream NHEJ factors to DSBs. Moreover, WASH role in DSB repair depends on its conserved C-terminal VCA domain and Arp2/3 activation. Our findings reveal a function and mechanistic insight for WASH in DNA DSB repair by the NHEJ pathway.

WASH interacts with Ku to regulate DNA double-stranded break repair.

Vacuolar protein sorting 35 (VPS35) is a major component of the retromer complex that regulates endosomal trafficking in eukaryotic cells. Recent studies have shown that VPS35 promotes tumor cell proliferation and affects the nuclear accumulation of its interacting partner. In this study, isobaric tags for relative and absolute quantitation (iTRAQ)-based mass spectrometry were used to measure the changes in nuclear protein abundance in VPS35-depleted HeLa cells. A total of 47 differentially expressed proteins were identified, including 27 downregulated and 20 upregulated proteins. Gene ontology (GO) analysis showed that the downregulated proteins included several minichromosome maintenance (MCM) proteins described as cell proliferation markers, and these proteins were present in the MCM2-7 complex, which is essential for DNA replication. Moreover, we validated that loss of VPS35 reduced the mRNA and protein expression of MCM2-7 genes. Notably, re-expression of VPS35 in VPS35 knockout HeLa cells rescued the expression of these genes. Functionally, we showed that VPS35 contributes to cell proliferation and maintenance of genomic stability of HeLa cells. Therefore, these findings reveal that VPS35 is involved in the regulation of MCM2-7 gene expression and establish a link between VPS35 and cell proliferation.

ITRAQ-based quantitative proteomic analysis reveals that VPS35 promotes the expression of MCM2-7 genes in HeLa cells.

WASHC1 is a member of the Wiskott-Aldrich syndrome protein (WASP) family and is involved in endosomal protein sorting and trafficking through the generation of filamentous actin (F-actin) via activation of the Arp2/3 complex. There is increasing evidence that WASHC1 is present in the nucleus and nuclear WASHC1 plays important roles in regulating gene transcription, DNA repair as well as maintaining nuclear organization. However, the multi-faceted functions of nuclear WASHC1 still need to be clarified.

WASHC1 interacts with MCM2-7 complex to promote cell survival under replication stress.

Cytoplasmic FAM21 works as a guiding protein in Wiskott-Aldrich Syndrome Protein and SCAR Homolog (WASH) complex by linking WASH complex to endosomes through its interaction with retromer. Recently, we have reported that nuclear WASH localizes to DNA double strand break (DSB) sites to promote DNA repair through non-homologous end-joining (NHEJ). However, whether FAM21, the close partner of WASH, is involved in the nuclear WASH localization and DNA repair remains to be clarified. Here, we show that FAM21 interacts with Ku and the interaction between C-terminal FAM21 and Ku is essential for its recruitment to DSB sites. Moreover, FAM21 depletion led to decreases in WASH recruitment to damaged DNA and repair capacity upon DNA damage. Taken together, these results reveal that FAM21 promotes DNA repair by orchestrating the recruitment of WASH to DSB sites, providing a mechanistic insight into WASH-dependent DNA DSB repair.

Xian Hong

Laboratory of Protein Structure and Function,
Institute of Medicine and Pharmacy,
Laboratory of Protein Structure and Function, Institute of Medicine and Pharmacy

Qiqihar Medical University

Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells Using GFP-Based Reporter Systems

Xian Hong

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory of Protein Structure and Function,Institute of Medicine and Pharmacy,Laboratory of Protein Structure and Function, Institute of Medicine and Pharmacy

Qiqihar Medical University

Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells Using GFP-Based Reporter Systems

Laboratory of Protein Structure and Function,
Institute of Medicine and Pharmacy,
Laboratory of Protein Structure and Function, Institute of Medicine and Pharmacy