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Mutational phospho-mimicry reveals a regulatory role for the XRCC4 and XLF C-terminal tails in modulating DNA bridging during classical non-homologous end joining.

eLife | 2017

XRCC4 and DNA Ligase 4 (LIG4) form a tight complex that provides DNA ligase activity for classical non-homologous end joining (the predominant DNA double-strand break repair pathway in higher eukaryotes) and is stimulated by XLF. Independently of LIG4, XLF also associates with XRCC4 to form filaments that bridge DNA. These XRCC4/XLF complexes rapidly load and connect broken DNA, thereby stimulating intermolecular ligation. XRCC4 and XLF both include disordered C-terminal tails that are functionally dispensable in isolation but are phosphorylated in response to DNA damage by DNA-PK and/or ATM. Here we concomitantly modify the tails of XRCC4 and XLF by substituting fourteen previously identified phosphorylation sites with either alanine or aspartate residues. These phospho-blocking and -mimicking mutations impact both the stability and DNA bridging capacity of XRCC4/XLF complexes, but without affecting their ability to stimulate LIG4 activity. Implicit in this finding is that phosphorylation may regulate DNA bridging by XRCC4/XLF filaments.

Pubmed ID: 28500754 RIS Download

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Non-profit plasmid repository dedicated to helping scientists around the world share high-quality plasmids. Facilitates archiving and distributing DNA-based research reagents and associated data to scientists worldwide. Repository contains over 65,000 plasmids, including special collections on CRISPR, fluorescent proteins, and ready-to-use viral preparations. There is no cost for scientists to deposit plasmids, which saves time and money associated with shipping plasmids themselves. All plasmids are fully sequenced for validation and sequencing data is openly available. We handle the appropriate Material Transfer Agreements (MTA) with institutions, facilitating open exchange and offering intellectual property and liability protection for depositing scientists. Furthermore, we curate free educational resources for the scientific community including a blog, eBooks, video protocols, and detailed molecular biology resources.

View all literature mentions

Addgene (data or information resource)

RRID:SCR_002037

Non-profit plasmid repository dedicated to helping scientists around the world share high-quality plasmids. Facilitates archiving and distributing DNA-based research reagents and associated data to scientists worldwide. Repository contains over 65,000 plasmids, including special collections on CRISPR, fluorescent proteins, and ready-to-use viral preparations. There is no cost for scientists to deposit plasmids, which saves time and money associated with shipping plasmids themselves. All plasmids are fully sequenced for validation and sequencing data is openly available. We handle the appropriate Material Transfer Agreements (MTA) with institutions, facilitating open exchange and offering intellectual property and liability protection for depositing scientists. Furthermore, we curate free educational resources for the scientific community including a blog, eBooks, video protocols, and detailed molecular biology resources.

View all literature mentions

RABBIT ANTI HUMAN XRCC4 (antibody)

RRID:AB_2218607

This polyclonal targets XRCC4

View all literature mentions

XLF antibody (antibody)

RRID:AB_778945

This polyclonal targets Human XLF

View all literature mentions

Anti-β-Actin Antibody (antibody)

RRID:AB_476744

This monoclonal targets slightly modified β-cytoplasmic actin N-terminal peptide, Ac-Asp-Asp-Asp-Ile-Ala-Ala-Leu-Val-Ile-Asp-Asn-Gly-Ser-Gly-Lys, conjugated to KLH

View all literature mentions

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RRID:CVCL_K253

Cell line XR-1 is a Spontaneously immortalized cell line with a species of origin Cricetulus griseus

View all literature mentions

HEK293T (cell line)

RRID:CVCL_0063

Cell line HEK293T is a Transformed cell line with a species of origin Homo sapiens (Human)

View all literature mentions

Microcal Origin (software resource)

RRID:SCR_002815

A complete graphing and data analysis software package that provides a suite of features catering to the needs of scientists and engineers. Main features: * Drag-and-drop import of data and images * Save import settings to a worksheet or external files for repeated use * Use saved settings to re-import files with a single click * Post-process imported data * Import Wizard with visual feedback * Handle non-standard files by programming

View all literature mentions

HCT 116 NHEJ1(-/-) (cell line)

RRID:CVCL_HD86

Cell line HCT 116 NHEJ1(-/-) is a Cancer cell line with a species of origin Homo sapiens (Human)

View all literature mentions

HCT 116 (cell line)

RRID:CVCL_0291

Cell line HCT 116 is a Cancer cell line with a species of origin Homo sapiens (Human)

View all literature mentions