PHF11 promotes DSB resection, ATR signaling, and HR

Abstract

Resection of double-strand breaks (DSBs) plays a critical role in their detection and appropriate repair. The 3 ′ ssDNA protrusion formed through resection activates the ATR-dependent DNA damage response (DDR) and is required for DSB repair by homologous recombination (HR). Here we report that PHF11 (plant homeodomain finger 11) encodes a previously unknown DDR factor involved in 5 ′ end resection, ATR signaling, and HR. PHF11 was identified based on its association with deprotected telomeres and localized to sites of DNA damage in S phase. Depletion of PHF11 diminished the ATR signaling response to telomere dysfunction and genome-wide DNA damage, reduced end resection at sites of DNA damage, resulted in compromised HR and misrejoining of S-phase DSBs, and increased the sensitivity to DNA-damaging agents. PHF11 interacted with the ssDNA-binding protein RPA and was found in a complex with several nucleases, including the 5 ′ dsDNA exonuclease EXO1. Biochemical experiments demonstrated that PHF11 stimulates EXO1 by overcoming its inhibition by RPA, suggesting that PHF11 acts (in part) by promoting 5 ′ end resection at RPA-bound sites of DNA damage. These findings reveal a role for PHF11 in DSB resection, DNA damage signaling, and DSB repair.[Keywords: PHF11; EXO1; RPA; ATR; DSB; resection; homologous recombination] Supplemental material is available for this article.Received October 9, 2016; revised version accepted December 22, 2016.Nuclear double-strand breaks (DSBs) activate the ATM and ATR kinase-dependent DNA damage response (DDR) pathways (for review, see Ciccia and Elledge 2010). Whereas the ATM kinase responds to the presence of dsDNA ends, activation of the ATR kinase requires the presence of ssDNA that is bound by RPA. In addition, activation of the ATR kinase requires the loading of the 9-1-1 complex on the double-stranded-single-stranded junction formed after 5 ′ end resection. Resection of the 5 ′ end of DSBs is therefore a critical step in the activation of ATR signaling. DSB resection is also required for the initiation of several DNA repair pathways, including homologous recombination (HR) and single-strand annealing (SSA) (for review, see Symington and Gautier 2011). HR can restore the original DNA sequence using the sister chromatid as a template for repair. After 5 ′ end resection, HR is initiated by the BRCA2-mediated loading of the Rad51 recombinase onto the ssDNA. In the absence of HR, S-phase DSBs can become a substrate for more error-prone DSB repair, including SSA and classical or alternative nonhomologous end-joining (NHEJ). DSB resection is initiated through the agency of BRCA1, the MRE11/RAD50/NBS1 (MRN) complex, and CtIP (for review, see Cejka 2015). The outcome is a ssDNA end with a short (∼20-nucleotide [nt]) 3 ′ overhang that is a substrate for further nucleolytic attack by EXO1, a dsDNA exonuclease that degrades only the 5 ′ strand to leave a 3 ′ overhang that can be thousands of nucleotides in length. In addition, an overlapping pathway involving the DNA2 nuclease acting in conjun...

The essential kinase ATR: ensuring faithful duplication of a challenging genome

“…For example, resection of a double-strand break 59,60 or deprotection of a telomere can activate ATR 10 . Also, some repair-related DNA processing such as that taking place during excision repair may be sufficient to generate a transient ATR-activating DNA structure, especially if repair is not completed promptly 61 .…”

Putting PHDs to work: PHF11 clears the way for EXO1 in double-strand break repair

“…The work of Gong et al (2017) thus begins a new chapter in which the motif of RPA mediation is expounded, demonstrating the universality of dealing with RPA during each step of HR. As noted above, PHF11 also interacts with several other repair factors, and it is still an open question as to the relative importance of its RPA displacement activities in comparison with its interactions with these myriad DNA repair and resection factors.…”

“…However, the precise choreography by which the 5 ′ resection apparatus circumvents RPA-mediated blockades in vivo stands as an open question. In this issue of Genes & Development, Gong et al (2017) identified plant homeodomain (PHD) finger 11 (PHF11) at uncapped telomeres by the proteomics of isolated chromatin segments (PICh) technique (Déjardin and Kingston 2009) and subsequently demonstrated an essential role for this new DDR factor in 5 ′ resection. PHF11 was described previously as a likely transcription factor gene adjacent to the IgE locus and associated with asthma and other inflammatory conditions (Zhang et al 2003).…”

“…C-terminal to this is the PHD finger motif, where two additional zinc ions bind (Liu et al 2014). Interestingly, of the DDR binding partners identified by Gong et al (2017), only RPA required the PHD finger to interact with PHF11. EXO1, the MRE11-RAD50-NBS1 complex, BARD1, and DNA2 could be pulled down by a PHF11 deletion that lacked the PHD finger but retained the pre-PHD region.…”

“…Might the PHD fingers of the AtBRCA1-AtBARD1 heterodimer function like PHF11 during 5 ′ resection in plants, thus hinting at a conserved reaction mechanism? Although the reported interaction between BRCA1-BARD1 and PHF11 will require further investigation to evaluate this insight, the study from Gong et al (2017) demonstrates clearly that PHD fingers are not unique to the DSB repair complexes of plants. The necessity to mediate RPA during resection emerges from the current work, which was not altogether unexpected considering that RPA negotiation is fundamental at other steps of HR (Fig.…”