Loading of the Nonhomologous End Joining Factor, Ku, on Protein-occluded DNA Ends

Abstract

The nonhomologous end joining pathway for DNA double strand break repair requires Ku to bind DNA ends and subsequently recruit other nonhomologous end joining factors, including the DNA-dependent protein kinase catalytic subunit and the XRCC4-Ligase IV complex, to the break site. Ku loads at a break by threading the DNA ends through a circular channel in its structure. This binding mechanism explains both the high specificity of Ku for ends and its ability to translocate along DNA once loaded. However, DNA in cells is typically coated with other proteins (e.g. histones), which might be expected to block the ability of Ku to load in this manner. Here we address how the nature of a protein obstruction dictates how Ku interacts with a DNA end. Ku is unable to access the ends within an important intermediate in V(D)J recombination (a complex of RAG proteins bound to cleaved recombination targeting signals), but Ku readily displaces the linker histone, H1, from DNA. Ku also retains physiological affinity for nucleosome-associated ends. Loading onto nucleosome-associated ends still occurs by threading the end through its channel, but rather than displacing the nucleosome, Ku peels as much as 50 bp of DNA away from the histone octamer surface. We suggest a model where Ku utilizes an unusual characteristic of its three-dimensional structure to recognize certain protein-occluded ends without the extensive remodeling of chromatin structure required by other DNA repair pathways. DNA double strand breaks (DSBs)2 pose a threat to genomic integrity. Nonhomologous end joining (NHEJ) is a major pathway for repair of exogenously introduced DSBs in mammals and is the only efficient way to repair DSB intermediates in V(D)J recombination (1). NHEJ requires the DNA binding heterodimer Ku to first recognize broken DNA ends (2-5) and subsequently recruit the additional NHEJ factors necessary to complete repair (e.g. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) (6), the XRCC4-LigaseIV complex (7,8), and polymerases (9, 10)). Consequently, deficiency in Ku cripples NHEJ and leads to severe immunodeficiency (11, 12) and cellular sensitivity to agents that cause DSBs (e.g. ionizing radiation) (13,14). The ability of Ku to recognize DNA ends requires the end to be inserted through a channel in its structure (5, 15, 16). This manner of binding makes Ku highly specific for DNA ends but requires that the ends must be accessible through 360°. In cells, DNA is generally coated with proteins (e.g. histones and other chromatin-associated proteins) that might be expected to block the ability of Ku to load on DNA ends and thus impair NHEJ.Here we have shown that Ku displays a variety of responses to protein obstructions at DNA ends. Ku is unable to bind a class of RAG (recombination activating gene) protein-bound DNA ends generated during V(D)J recombination. In contrast, Ku can displace certain proteins (e.g. histone H1) from DNA and retains the ability to load on nucleosome-associated ends by peeling up to 50 bp of DNA away from the ...

The Ku heterodimer: Function in DNA repair and beyond

“…The next step, aided by the recruitment of Rad52, is the invasion of this filament strand into the DNA of the homologous chromosome to form a D-loop structure. A polymerase extends the 3 0 overhang to create a cross structure known as the Holliday that only one or two Ku molecules were able to load onto a chromatin substrate [56]. A recently developed method for visualizing Ku foci also demonstrated that on average there are only two Ku molecules present at a DSB in vivo, presumably one at each end of the DNA break [57].…”

Yeast HMO1: Linker Histone Reinvented

“…To efficiently repair DSBs by Rad51p-and Rad54p-mediated homologous recombination, the linker histone H1 is evicted by a histone chaperone, Nap1p, from a reconstituted nucleosome array, suggesting that eviction of H1 promotes repair by homologous recombination (86). In contrast, Ku, which is integral to DSB repair by nonhomologous end joining, has been reported to readily displace human H1 from DNA ends (87). In human cells, ubiquitylation of H1 at DSB sites was also recently implicated in the recruitment of repair factors, adding to the collection of histone marks that contribute to repair events (88).…”

MgLig4, a homolog of Neurospora crassa Mus-53 (DNA ligase IV), is involved in, but not essential for, non-homologous end-joining events in Magnaporthe grisea

“…In many eukaryotes, including humans, plants, insects, and filamentous fungi, the NHEJ system appears to be the main DSBs repair pathway (Pastink et al, 2001). In the mammalian NHEJ system, DSBs repair involve the detection and binding of the DSB ends by Ku70/Ku80 heterodimers followed by the gathering of additional NHEJ factors, including the DNA-dependent protein kinase (DNA-PK), DNA ligase IV-Xrcc4 complex, and polymerases to the DSBs ends to complete the repair (Roberts and Ramsden, 2007). Recently, DNA ligase III was identified as a component of a backup pathway to the DNA-PK/ DNA ligase IV dependent NHEJ system in mammalian cells (Wang et al, 2006).…”