Have you seen ? Rad 17 , the clamp loader that loads more than clamps

T he cellular response to DNA doublestrand breaks (DSBs) involves immediate binding of break sites by early-acting DNA repair and checkpoint proteins (Goodarzi & Jeggo, 2013). Coordinated loading of DNA end processing factors promotes repair through either homologous or non-homologous mechanisms, and signaling of breaks through post-translational modifications that emanate from the break site ensures rapid cell cycle arrest or apoptosis. One of the first complexes arriving at a break to mediate these events is the Mre11-Rad50-Nbs1 (MRN) complex, which promotes repair of breaks through 50 strand resection and homologous recombination and also initiates a signaling cascade through the ATM protein kinase to trigger checkpoint activation. It has been understood for some time that MRN is recruited to sites of DNA breaks via at least two different mechanisms. Over a time course of DNA damage (ionizing radiation in most cases), MRN is observed by indirect immunofluorescence to arrive within seconds of double-strand break induction (early recruitment) and also remains at a subset of these sites in distinct foci at later time points (retention) (Lukas et al, 2004). The retention of MRN at regions of chromatin adjacent to break sites is much better understood: MRN binds directly to the Mdc1 protein through phospho-specific interactions between the Nbs1 component of MRN and the constitutively phosphorylated Mdc1 protein, which is recruited to chromatin near DSBs through recognition of the phosphorylated form of the histone H2AX (c-H2AX) (Goodarzi & Jeggo, 2013). Cells deleted for H2AX still exhibit early recruitment of MRN but are deficient in forming the large foci at later time points that are dependent on Mdc1 (Celeste et al, 2003). This distinction between two kinds of binding patterns and kinetics was also observed in the physical size of the c-H2AXindependent Nbs1 foci which were limited to a smaller region compared to c-H2AX foci and thus termed “microfoci” in the absence of the Mdc1-dependent late retention pathway (Lukas et al, 2004). We have assumed that early recruitment of MRN to the actual break site (as opposed to the mega-basesized region of the chromatin surrounding the break site) is mediated by MRN itself, since the complex exhibits picomolar affinity for DNA ends, and in vitro reconsitituted assays for DNA binding, 50 resection, end tethering, and ATM activation do not require any other factors (Lee & Paull, 2007). However, a new study in this issue of The EMBO Journal shows that the Rad17 protein, previously implicated in ATR signaling through its role as part of the Rad17-Rfc2-5 clamp loader for the Rad1Rad9-Hus1 (9-1-1) complex, is necessary for the early recruitment of MRN to doublestrand break sites (Wang et al, 2014). In this article, Wang and colleagues show that depletion of Rad17 causes a dramatic loss of ionizing radiation (IR)-induced foci including c-H2AX, Nbs1, Mdc1, and RPA as well as defects in ATM-dependent phosphorylation and homologous recombination, suggesting that Rad17 has a role in the early stages of DNA DSB response (Wang et al, 2014). Previous studies using genetic deletions of Rad17 had suggested a role for this factor in homologous recombination (Nishino et al, 2008; Saberi et al, 2008), although the function of Rad17 in this process was not clear. Here, the authors show that Rad17 is required for the early recruitment of the MRN complex to DSB sites but is not required for Mdc1/c-H2AX-dependent recruitment of the MRN complex at late time points after IR. A direct interaction between Nbs1 and Rad17 was demonstrated, which requires ATM phosphorylation of Rad17 on Thr622. They also show that expression of a T622A Rad17 mutant causes severe reduction in MRN complex levels on chromatin after IR treatment and loss of ATM, Nbs1, and Chk2 phosphorylation at early time points after IR treatment. Taken together, these data suggest that Rad17 has a role in the recruitment of the MRN complex to DSBs independent of Mdc1/c-H2AX (Fig 1). The MRN complex has two important roles after DNA damage: initiation of DNA repair and activation of ATM. The MRN complex exhibits endoand exo-nuclease activity on double-stranded DNA and interacts with the CtIP endonuclease, promoting homologous recombination by initiating DSB end resection (Goodarzi & Jeggo, 2013). In the article by Wang et al, the authors show that Rad17 recruits the MRN complex