ZMYM3 regulates BRCA1 localization at damaged chromatin to promote DNA repair.

Chromatin connects DNA damage response factors to sites of damaged DNA to promote the signaling and repair of DNA lesions. The histone H2A variants H2AX, H2AZ, and macroH2A represent key chromatin constituents that facilitate DNA repair. Through proteomic screening of these variants, we identified ZMYM3 (zinc finger, myeloproliferative, and mental retardation-type 3) as a chromatin-interacting protein that promotes DNA repair by homologous recombination (HR). ZMYM3 is recruited to DNA double-strand breaks through bivalent interactions with both histone and DNA components of the nucleosome. We show that ZMYM3 links the HR factor BRCA1 to damaged chromatin through specific interactions with components of the BRCA1-A subcomplex, including ABRA1 and RAP80. By regulating ABRA1 recruitment to damaged chromatin, ZMYM3 facilitates the fine-tuning of BRCA1 interactions with DNA damage sites and chromatin. Consistent with a role in regulating BRCA1 function, ZMYM3 deficiency results in impaired HR repair and genome instability. Thus, our work identifies a critical chromatin-binding DNA damage response factor, ZMYM3, which modulates BRCA1 functions within chromatin to ensure the maintenance of genome integrity.

CtIP: A DNA damage response protein at the intersection of DNA metabolism.

The mammalian CtIP protein and its orthologs in other eukaryotes promote the resection of DNA double-strand breaks and are essential for meiotic recombination. Here we review the current literature supporting the role of CtIP in DNA end processing and the importance of CtIP endonuclease activity in DNA repair. We also examine the regulation of CtIP function by post-translational modifications, and its involvement in transcription- and replication-dependent functions through association with other protein complexes. The tumor suppressor function of CtIP likely is dependent on a combination of these roles in many aspects of DNA metabolism.

Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1.

DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). The C terminal binding protein-interacting protein (CtIP) is phosphorylated in G2 by cyclin-dependent kinases to initiate resection and promote HR. CtIP also exerts functions during NHEJ, although the mechanism phosphorylating CtIP in G1 is unknown. In this paper, we identify Plk3 (Polo-like kinase 3) as a novel DSB response factor that phosphorylates CtIP in G1 in a damage-inducible manner and impacts on various cellular processes in G1. First, Plk3 and CtIP enhance the formation of ionizing radiation-induced translocations; second, they promote large-scale genomic deletions from restriction enzyme-induced DSBs; third, they are required for resection and repair of complex DSBs; and finally, they regulate alternative NHEJ processes in Ku(-/-) mutants. We show that mutating CtIP at S327 or T847 to nonphosphorylatable alanine phenocopies Plk3 or CtIP loss. Plk3 binds to CtIP phosphorylated at S327 via its Polo box domains, which is necessary for robust damage-induced CtIP phosphorylation at S327 and subsequent CtIP phosphorylation at T847.

RecR-mediated modulation of RecF dimer specificity for single- and double-stranded DNA.

RecF pathway proteins play an important role in the restart of stalled replication and DNA repair in prokaryotes. Following DNA damage, RecF, RecR, and RecO initiate homologous recombination (HR) by loading of the RecA recombinase on single-stranded (ss) DNA, protected by ssDNA-binding protein. The specific role of RecF in this process is not well understood. Previous studies have proposed that RecF directs the RecOR complex to boundaries of damaged DNA regions by recognizing single-stranded/double-stranded (ss/ds) DNA junctions. RecF belongs to ABC-type ATPases, which function through an ATP-dependent dimerization. Here, we demonstrate that the RecF of Deinococcus radiodurans interacts with DNA as an ATP-dependent dimer, and that the DNA binding and ATPase activity of RecF depend on both the structure of DNA substrate, and the presence of RecR. We found that RecR interacts as a tetramer with the RecF dimer. RecR increases the RecF affinity to dsDNA without stimulating ATP hydrolysis but destabilizes RecF binding to ssDNA and dimerization, likely due to increasing the ATPase rate. The DNA-dependent binding of RecR to the RecF-DNA complex occurs through specific protein-protein interactions without significant contributions from RecR-DNA interactions. Finally, RecF neither alone nor in complex with RecR preferentially binds to the ss/dsDNA junction. Our data suggest that the specificity of the RecFOR complex toward the boundaries of DNA damaged regions may result from a network of protein-protein and DNA-protein interactions, rather than a simple recognition of the ss/dsDNA junction by RecF.

Structural conservation of RecF and Rad50: implications for DNA recognition and RecF function.

RecF, together with RecO and RecR, belongs to a ubiquitous group of recombination mediators (RMs) that includes eukaryotic proteins such as Rad52 and BRCA2. RMs help maintain genome stability in the presence of DNA damage by loading RecA-like recombinases and displacing single-stranded DNA-binding proteins. Here, we present the crystal structure of RecF from Deinococcus radiodurans. RecF exhibits a high degree of structural similarity with the head domain of Rad50, but lacks its long coiled-coil region. The structural homology between RecF and Rad50 is extensive, encompassing the ATPase subdomain and the so-called 'Lobe II' subdomain of Rad50. The pronounced structural conservation between bacterial RecF and evolutionarily diverged eukaryotic Rad50 implies a conserved mechanism of DNA binding and recognition of the boundaries of double-stranded DNA regions. The RecF structure, mutagenesis of conserved motifs and ATP-dependent dimerization of RecF are discussed with respect to its role in promoting presynaptic complex formation at DNA damage sites.