Recruitment and Dissociation of Nonhomologous End Joining Proteins at a DNA Double-Strand Break in <i>Saccharomyces cerevisiae</i>

Wu, Dongliang; Topper, Leana M.; Wilson, Thomas E.

doi:10.1534/genetics.107.083535

Cited by 122 publications

(171 citation statements)

References 68 publications

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“…5C), suggesting that interactions with both yKu and Nej1 contribute to enhanced DNA binding by Dnl4-Lif1. Our data and other published studies (19,24) are consistent with a model in which yKu initiates binding at a DSB and then independently recruits Nej1 and Dnl4-Lif1. To mimic this scenario, we examined the effect of mixing Nej1 and Dnl4-Lif1 prior to loading onto DNA bound by yKu.…”

Section: Journal Of Biological Chemistry 4935supporting

confidence: 92%

“…Vivo DSBs-Although Nej1 is a direct participant in yeast NHEJ (24), the mechanisms by which it contributes to NHEJ are not well defined. In accord with published studies (20 -23), deletion of NEJ1 in the SLY1 genetic background resulted in a defect in NHEJ, measured either by growth when HO endonuclease was constitutively expressed (Fig.…”

Section: Nej1 Is Recruited By and Stabilizes The Binding Of Yku To Inmentioning

confidence: 99%

“…There is now compelling evidence that Nej1 is a direct participant in NHEJ, in addition to regulating the subcellular localization of Dnl4-Lif1 (24). This notion is supported by parallel studies with the mammalian ortholog of Nej1, XLF (XRCC4-like factor; Cernunnos) (25)(26)(27).…”

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confidence: 94%

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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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Section: Journal Of Biological Chemistry 4935supporting

confidence: 92%

Section: Nej1 Is Recruited By and Stabilizes The Binding Of Yku To Inmentioning

confidence: 99%

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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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“…The HO(12) suicide deletion strain YW1276 [MATa-inc, ade2THOSD(11) TSTE3-MET15, his3D1, leu2D, met15D, ura3D] was previously described . The chromatin immunoprecipitation (ChIP) strain YW1752 (MATa-incTChIP-QPCRT URA3, can1DTrepair-QPCR, DNL4-Myc13THisMX6, gal1T HOcsTHOcod, his3D1, leu2D0, met15D0, ura3D) was previously described (Wu et al 2008). Both YW1276 and YW1752 are direct descendants of BY4741/BY4742.…”

Section: Methodsmentioning

confidence: 99%

Recruitment of Saccharomyces cerevisiae Dnl4–Lif1 Complex to a Double-Strand Break Requires Interactions With Yku80 and the Xrs2 FHA Domain

Palmbos

Daley³

et al. 2008

Genetics

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Nonhomologous end joining (NHEJ) in yeast depends on eight different proteins in at least three different functional complexes: Yku70-Yku80 (Ku), Dnl4-Lif1-Nej1 (DNA ligase IV), and Mre11-Rad50-Xrs2 (MRX). Interactions between these complexes at DNA double-strand breaks (DSBs) are poorly understood but critical for the completion of repair. We previously identified two such contacts that are redundantly required for NHEJ, one between Dnl4 and the C terminus of Yku80 and one between the forkhead-associated (FHA) domain of Xrs2 and the C terminus of Lif1. Here, we first show that mutation of the Yku80 C terminus did not impair Ku binding to DSBs, supporting specificity of the mutant defect to the ligase interaction. We next show that the Xrs2-Lif1 interaction depends on Xrs2 FHA residues (R32, S47, R48, and K75) analogous to those known in other proteins to contact phosphorylated threonines. Two potential target threonines in Lif1 (T417 and T387) were inferred by identifying regions similar to a site in the human Lif1 homolog, XRCC4, known to be bound by the FHA domain of polynucleotide kinase. Mutating these threonines, especially T417, abolished the Xrs2-Lif1 interaction and impaired NHEJ epistatically with Xrs2 FHA mutation. Combining mutations that selectively disable the Yku80-Dnl4 and Xrs2-Lif1 interactions abrogated both NHEJ and DNA ligase IV recruitment to a DSB. The collected results indicate that the Xrs-Lif1 and Yku80-Dnl4 interactions are important for formation of a productive ligase-DSB intermediate.

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“…Genetic experiments in yeast have shown that Ku occludes DNA ends from Exo1. Ku and MRX/MRN are among the first proteins recruited to DSBs (91,92). Importantly, the MRX nuclease activity and Sae2 (orthologous to CtIP) function in conjunction to remove Ku (92)(93)(94)(95).…”

Section: Removal Of Ku From Dsb Endsmentioning

confidence: 99%

53BP1, BRCA1, and the Choice between Recombination and End Joining at DNA Double-Strand Breaks

2014

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When DNA double-strand breaks occur, the cell cycle stage has a major influence on the choice of the repair pathway employed. Specifically, nonhomologous end joining is the predominant mechanism used in the G 1 phase of the cell cycle, while homologous recombination becomes fully activated in S phase. Studies over the past 2 decades have revealed that the aberrant joining of replication-associated breaks leads to catastrophic genome rearrangements, revealing an important role of DNA break repair pathway choice in the preservation of genome integrity. 53BP1, first identified as a DNA damage checkpoint protein, and BRCA1, a well-known breast cancer tumor suppressor, are at the center of this choice. Research on how these proteins function at the DNA break site has advanced rapidly in the recent past. Here, we review what is known regarding how the repair pathway choice is made, including the mechanisms that govern the recruitment of each critical factor, and how the cell transitions from end joining in G 1 to homologous recombination in S/G 2 .

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Recruitment and Dissociation of Nonhomologous End Joining Proteins at a DNA Double-Strand Break in Saccharomyces cerevisiae

Cited by 122 publications

References 68 publications

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

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

Recruitment of Saccharomyces cerevisiae Dnl4–Lif1 Complex to a Double-Strand Break Requires Interactions With Yku80 and the Xrs2 FHA Domain

53BP1, BRCA1, and the Choice between Recombination and End Joining at DNA Double-Strand Breaks

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