Cernunnos/XLF promotes the ligation of mismatched and noncohesive DNA ends

Tsai, Chun J.; Kim, Sunny A.; Chu, Gilbert

doi:10.1073/pnas.0702620104

Cited by 167 publications

(169 citation statements)

References 25 publications

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“…The crystal structure of the L4-X4-Cernunnos complex with the fulllength proteins should help to clarify the binding mode of the complete ligation complex. DSB repair studies in the presence or absence of Cernunnos have shown that Cernunnos promotes DNA end ligation by the L4-X4 complex (25,33). From our observation, we propose that the oligomerization of the X4-Cernunnos complex could be a mechanism to facilitate the recruitment of two or more L4-X4 complexes to DNA DSB repair points (Fig.…”

Section: Discussionmentioning

confidence: 77%

“…The association between Cernunnos and L4-X4 complex is mediated primarily through an interaction with X4 (14), although a weak interaction with L4 has also been reported (16). Cernunnos stimulates the ligase activity of the L4-X4 complex (20,21,(23)(24)(25) and seems particularly important for the ligation of mismatched or noncohesive DNA ends (25,26). Like X4, Cernunnos is a homodimer (27,28).…”

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confidence: 99%

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Structural characterization of filaments formed by human Xrcc4–Cernunnos/XLF complex involved in nonhomologous DNA end-joining

Ropars

Drevet

Legrand

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

123

129

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Cernunnos/XLF is a core protein of the nonhomologous DNA end-joining (NHEJ) pathway that processes the majority of DNA double-strand breaks in mammals. Cernunnos stimulates the final ligation step catalyzed by the complex between DNA ligase IV and Xrcc4 (X4). Here we present the crystal structure of the X4 1-157 -Cernunnos 1-224 complex at 5.5-Å resolution and identify the relative positions of the two factors and their binding sites. The X-ray structure reveals a filament arrangement for X4 1-157 and Cernunnos 1-224 homodimers mediated by repeated interactions through their N-terminal head domains. A filament arrangement of the X4-Cernunnos complex was confirmed by transmission electron microscopy analyses both with truncated and full-length proteins. We further modeled the interface and used structure-based site-directed mutagenesis and calorimetry to characterize the roles of various residues at the X4-Cernunnos interface. We identified four X4 residues (Glu 55 , Asp 58 , Met 61 , and Phe 106 ) essential for the interaction with Cernunnos. These findings provide new insights into the molecular bases for stimulatory and bridging roles of Cernunnos in the final DNA ligation step.D NA double-strand breaks (DSBs) are the most toxic DNA lesions in the genome, and unrepaired DSBs can cause large-scale losses of genetic information through chromosome rearrangement (1, 2). These DNA damages result from exposure to exogenous damaging agents, such as ionizing radiation, radiomimetic compounds, and topoisomerase inhibitors. DSBs are also obligate intermediates in several recombination processes in vertebrates, including antigen receptor gene rearrangement, V(D)J recombination (3, 4). In higher eukaryotes, DSBs are repaired by several mechanisms, among which nonhomologous end-joining (NHEJ) represents the major pathway, particularly when sister chromatids are not available (5). Deficiency in the NHEJ machinery results in sensitivity to ionizing radiation and severe combined immune deficiencies in humans and mice due to abortive V(D)J recombination (6). NHEJ is orchestrated by at least seven proteins. The Ku70/Ku80 heterodimer adopts a preformed ring-shaped structure that recognizes and encircles the duplex DNA ends at the DSB (7). Ku70/Ku80 recruits a 469-kDa serine/threonine protein kinase, the DNA-PK catalytic subunit (DNA-PKcs), via a direct interaction and shifts about 10 bp inward so that DNA-PKcs acquires a position at the terminus through its large open-ring cradle structure (8). Upon association with Ku and DNA, DNA-PKcs is activated and phosphorylates several proteins including itself and Ku70/Ku80. The DNA-PK holoenzyme, constituted by Ku and DNA-PKcs, plays a central role in NHEJ. Among other functions, DNA-PK mediates the end-bridging of the DSB extremities (9), regulates access to the DNA ends by processing enzymes such as the DNA-PKcs-associated Artemis nuclease (10, 11), and recruits the Xrcc4-ligase IV complex to DNA ends for the ligation step (12). The Xrcc4-ligase IV complex carries out the final joinin...

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Section: Discussionmentioning

confidence: 77%

mentioning

confidence: 99%

Structural characterization of filaments formed by human Xrcc4–Cernunnos/XLF complex involved in nonhomologous DNA end-joining

Ropars

Drevet

Legrand

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

123

129

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“…These proteins are structurally similar to and interact with the yeast Lif1 and human XRCC4 subunit of the DNA ligase IV complex (20 -23, 25, 28, 30, 35). In addition, XLF enhances joining by DNA ligase IV-XRCC4 of both matched and mismatched DNA ends (31)(32)(33)(34)46). Using a combination of molecular genetic and biochemical approaches, we have demonstrated that Nej1 not only contributes to the final ligation step of NHEJ but also plays a critical role in the initiation of the NHEJ pathway.…”

Section: Discussionmentioning

confidence: 95%

Yeast Nej1 Is a Key Participant in the Initial End Binding and Final Ligation Steps of Nonhomologous End Joining

Chen¹,

Tomkinson

2011

Journal of Biological Chemistry

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In Saccharomyces cerevisiae, the key components of the nonhomologous end joining (NHEJ) pathway that repairs DNA double-strand breaks (DSBs) are yeast Ku (yKu), Mre11-Rad50-Xrs2, Dnl4-Lif1, and Nej1. Here, we examined the role of Nej1 in NHEJ by a combination of molecular genetic and biochemical approaches. As expected, the recruitment of Nej1 to in vivo DSBs is dependent upon yKu. Surprisingly, Nej1 is required for the stable binding of yKu to in vivo DSBs, in addition to Dnl4-Lif1. Thus, Nej1 and Dnl4-Lif1 are independently recruited by yKu to in vivo DSBs, forming a stable ternary complex that channels DSBs into the NHEJ pathway. In accord with these results, purified Nej1 interacts with yKu and preferentially binds to DNA ends bound by yKu. Furthermore, the binding of a mixture of Nej1 and Dnl4-Lif1 to DNA ends bound by yKu is greater than the sum of the binding of the individual proteins, indicating that pairwise interactions among yKu, Nej1, and Dnl4-Lif1 contribute to complex assembly at DNA ends. Nej1 stimulates intermolecular ligation by Dnl4-Lif1, but, more interestingly, the addition of Nej1 results in more than one intermolecular ligation per Dnl4 molecule. Thus, Nej1 not only plays an important role in determining repair pathway choice by participating in the initial NHEJ complex formed at DSBs but also contributes to the reactivation of Dnl4-Lif1 after repair is complete, thereby increasing the capacity of the NHEJ repair pathway.

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“…The gene was cloned independently from immunodeficient patients that showed radio sensitivity (Buck et al, 2006) and by a yeast 2-hybrid screen for proteins that interact with XRCC4 (Ahnesorg et al, 2006). It stimulates NHEJ, which appears to be mainly important for noncomplementary DNA ends, but does not seem to be an absolute requirement for the majority of DSBs (Gu et al, 2007;Tsai et al, 2007;Wu et al, 2007).…”

Section: Dna Ligase IV Xrcc4 and Xlf/cernunnosmentioning

confidence: 99%

Non-homologous end-joining, a sticky affair

2007

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Rejoining of broken chromosomes is crucial for cell survival and prevention of malignant transformation. Most mammalian cells rely primarily on the non-homologous end-joining pathway of DNA double-strand break (DSB) repair to accomplish this task. This review focuses both on the core non-homologous end-joining machinery, which consists of DNA-dependent protein kinase and the ligase IV/XRCC4 complex, and on accessory factors that facilitate rejoining of a subset of the DSBs. We discuss how the ATM protein kinase and the Mre11/Rad50/Nbs1 complex might function in DSB repair and what role ionizing radiation-induced foci may play in this process.

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Cernunnos/XLF promotes the ligation of mismatched and noncohesive DNA ends

Cited by 167 publications

References 25 publications

Structural characterization of filaments formed by human Xrcc4–Cernunnos/XLF complex involved in nonhomologous DNA end-joining

Structural characterization of filaments formed by human Xrcc4–Cernunnos/XLF complex involved in nonhomologous DNA end-joining

Yeast Nej1 Is a Key Participant in the Initial End Binding and Final Ligation Steps of Nonhomologous End Joining

Non-homologous end-joining, a sticky affair

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