title: Sliding sleeves of XRCC4-XLF bridge DNA and connect fragments of broken DNA.
abstract: Non-homologous end joining (NHEJ) is the primary pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells. Such breaks are formed, for example, during gene-segment rearrangements in the adaptive immune system or by cancer therapeutic agents. Although the core components of the NHEJ machinery are known, it has remained difficult to assess the specific roles of these components and the dynamics of bringing and holding the fragments of broken DNA together. The structurally similar XRCC4 and XLF proteins are proposed to assemble as highly dynamic filaments at (or near) DSBs. Here we show, using dual- and quadruple-trap optical tweezers combined with fluorescence microscopy, how human XRCC4, XLF and XRCC4-XLF complexes interact with DNA in real time. We find that XLF stimulates the binding of XRCC4 to DNA, forming heteromeric complexes that diffuse swiftly along the DNA. Moreover, we find that XRCC4-XLF complexes robustly bridge two independent DNA molecules and that these bridges are able to slide along the DNA. These observations suggest that XRCC4-XLF complexes form mobile sleeve-like structures around DNA that can reconnect the broken ends very rapidly and hold them together. Understanding the dynamics and regulation of this mechanism will lead to clarification of how NHEJ proteins are involved in generating chromosomal translocations.
authors.forename: Ineke
authors.initials: I
authors.lastname: Brouwer
authors.forename: Gerrit
authors.initials: G
authors.lastname: Sitters
authors.forename: Andrea
authors.initials: A
authors.lastname: Candelli
authors.forename: Stephanie J
authors.initials: SJ
authors.lastname: Heerema
authors.forename: Iddo
authors.initials: I
authors.lastname: Heller
authors.forename: Abinadabe J
authors.initials: AJ
authors.lastname: de Melo
authors.forename: Hongshan
authors.initials: H
authors.lastname: Zhang
authors.forename: Davide
authors.initials: D
authors.lastname: Normanno
authors.forename: Mauro
authors.initials: M
authors.lastname: Modesti
authors.forename: Erwin J G
authors.initials: EJ
authors.lastname: Peterman
authors.forename: Gijs J L
authors.initials: GJ
authors.lastname: Wuite
mesh_terms: D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D045643:DNA Repair Enzymes / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004058:Diffusion; D006801:Humans; D008856:Microscopy, Fluorescence; D009068:Movement; D052898:Optical Tweezers; D014178:Translocation, Genetic
chemical_list: D004268:DNA-Binding Proteins; C508551:NHEJ1 protein, human; C101467:XRCC4 protein, human; D045643:DNA Repair Enzymes