Academic journal article Genetics

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

Academic journal article Genetics

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

Article excerpt

ABSTRACT

Nonhomologous end joining (NHEJ) is an important DNA double-strand-break (DSB) repair pathway that requires three protein complexes in Saccharomyces cerevisiae: the Ku heterodimer (Yku70-Yku80), MRX (Mre11-Rad50-Xrs2), and DNA ligase IV (Dnl4-Lif1), as well as the ligase-associated protein Nej1. Here we use chromatin immunoprecipitation from yeast to dissect the recruitment and release of these protein complexes at HO-endonuclease-induced DSBs undergoing productive NHEJ. Results revealed that Ku and MRX assembled at a DSB independently and rapidly after DSB formation. Ligase IV appeared at the DSB later than Ku and MRX and in a strongly Ku-dependent manner. Ligase binding was extensive but slightly delayed in rad50 yeast. Ligase IV binding occurred independently of Nej1, but instead promoted loading of Nej1. Interestingly, dissociation of Ku and ligase from unrepaired DSBs depended on the presence of an intact MRX complex and ATP binding by Rad50, suggesting a possible role of MRX in terminating a NHEJ repair phase. This activity correlated with extended DSB resection, but limited degradation of DSB ends occurred even in MRX mutants with persistently bound Ku. These findings reveal the in vivo assembly of the NHEJ repair complex and shed light on the mechanisms controlling DSB repair pathway utilization.

NONHOMOLOGOUS end joining (NHEJ) is a principalmechanismfor repairing DNA double-strand breaks (DSBs) in which the two DSB ends are directly rejoined (Wilson 2007). As such, NHEJ is critical for maintaining genome stability. Many NHEJ proteins are known, but how they cooperate to execute repair in a living cell is poorly understood. In the model organism Saccharomyces cerevisiae, three major preformed protein complexes are required for all NHEJ reactions: Ku, the MRX complex, and DNA ligase IV (Dudasova et al. 2004; Daley et al. 2005). Yku70 and Yku80 formthe yeast Ku heterodimer, which by homology with human Ku is inferred to form a ring that binds DNA by sliding a DSB end through its opening (Walker et al. 2001). This binding is a principal means of DSB recognition that is critical for NHEJ, but, interestingly, unimportant for the competing homologous recombination (HR) pathway.

Mre11, Rad50, and Xrs2 (Nbs1 in mammalian cells) form the MRX complex (Usui et al. 1998), which also binds DNA but without a requirement for DSB ends as with Ku (Trujillo et al. 2003). Also unlike Ku, MRX plays a role in HR, possibly regulating repair pathway utilization through actions in 59 resection of DSB ends (Connelly and Leach 2002; Symington 2002). Rad50 is composed of two globular ATPase domains separated by a long coiled-coil region that self-associates at its end (Anderson et al. 2001; Wiltzius et al. 2005). Mre11 has an N-terminal nuclease domain, which binds near the Rad50 ATPase to create a DNA-binding head (Usui et al. 1998; Hopfner et al. 2001). Mre11 is also required for interaction between Rad50 and Xrs2 (Usui et al. 1998; Chen et al. 2001). Xrs2 harbors N-terminal FHA and BRCT domains and a C-terminal Mre11-binding domain (Shima et al. 2005) and is also required for efficient DNA binding by MRX (Trujillo et al. 2003).

Yeast DNA ligase IV is composed of Dnl4 (homologous to human Lig4) (Wilson et al. 1997) and Lif1 (XRCC4 in humans) (Herrmann et al. 1998). Dnl4 is a typical ATP-dependent DNA ligase with tandem Cterminal BRCT domains that interact with a coiled-coil region of Lif1 (Dore et al. 2006). This interaction is strong and physically stabilizes Dnl4 (Herrmann et al. 1998), but further actions of Lif1 are enigmatic. A third ligase-associated protein is Nej1 (XLF/Cernunnos in humans) (Revy et al. 2006), which plays a further poorly defined supporting role through less stable interactions with the globular head of Lif1 (Frank-Vaillant and Marcand 2001). Unlike Ku and MRX, which function in telomere maintenance (Boulton and Jackson 1998; Tsukamoto et al. 2001), and, in the case of MRX, HR andDNAdamage checkpoints (Sunget al. …

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