Phosphorylated CtIP Functions as a Co-factor of the MRE11-RAD50-NBS1 Endonuclease in DNA End Resection

Anand, Roopesh; Ranjha, Lepakshi; Cannavó, Elda; Ćejka, Petr

doi:10.1016/j.molcel.2016.10.017

Cited by 242 publications

(289 citation statements)

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“…The intrinsic 3'→5' exonuclease activity of the MRE11 component cannot generate these 3'-terminated overhangs by acting directly at a DNA end and relies on the C-terminal binding protein interacting protein (CtIP) to stimulate MRN endonuclease activity to incise distal from the break. Next, the 3' exonuclease activity can degrade DNA from the incision back towards the DNA end, thus creating the 3'-terminated ssDNA overhangs that can further undergo long range resection (e.g., by EXO1 or DNA2-BLM) (45,46). This processing may have implications for the binding of Ku to DNA ends since MRE11 endonuclease activity occurs upstream of the Ku-bound DNA end.…”

Section: Overview Of Nhej In Humans and Its Relationship With Other Pmentioning

confidence: 99%

Nonhomologous DNA end-joining for repair of DNA double-strand breaks

Pannunzio

Watanabe

Lieber

2018

Journal of Biological Chemistry

372

360

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Nonhomologous DNA end joining (NHEJ) is the predominant DSB repair pathway throughout the cell cycle and accounts for nearly all DSB repair outside of the S and G2 phases. NHEJ relies on Ku to thread onto DNA termini and thereby improve the affinity of the NHEJ enzymatic components consisting of polymerases (Pol m and Pol l), a nuclease (the Artemis·DNA-PKcs complex), and a ligase (XLF·XRCC4·Lig4 complex). Each of the enzymatic components is distinctive for its versatility in acting on diverse incompatible DNA end configurations coupled with a flexibility in loading order, resulting in many possible junctional outcomes from one DSB. DNA ends can either be directly ligated or, if the ends are incompatible, processed until a ligatable configuration is achieved that is often stabilized by up to 4 bp of terminal microhomology. Processing of DNA ends results in nucleotide loss or addition, explaining why DSBs repaired by NHEJ are rarely restored to their original DNA sequence. Thus, NHEJ is a single pathway with multiple enzymes at its disposal to repair DSBs, resulting in a diversity of repair outcomes.

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Section: Overview Of Nhej In Humans and Its Relationship With Other Pmentioning

confidence: 99%

Nonhomologous DNA end-joining for repair of DNA double-strand breaks

Pannunzio

Watanabe

Lieber

2018

Journal of Biological Chemistry

372

360

View full text Add to dashboard Cite

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“…Likewise, the catalytic function of CtIP has also been shown to be dispensable for resection of clean DSBs in human cells (43). Furthermore, in reconstituted reactions with purified proteins, Sae2–MRX or CtIP–MRN, prefer blocked ends over free ends for 5′ strand cleavage (41,42). Together, these observations suggest the possibility that resection of clean DSBs is initiated by a different mechanism.…”

Section: Introductionmentioning

confidence: 99%

Dna2 initiates resection at clean DNA double-strand breaks

Paudyal

Yan

et al. 2017

Nucleic Acids Research

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Nucleolytic resection of DNA double-strand breaks (DSBs) is essential for both checkpoint activation and homology-mediated repair; however, the precise mechanism of resection, especially the initiation step, remains incompletely understood. Resection of blocked ends with protein or chemical adducts is believed to be initiated by the MRN complex in conjunction with CtIP through internal cleavage of the 5′ strand DNA. However, it is not clear whether resection of clean DSBs with free ends is also initiated by the same mechanism. Using the Xenopus nuclear extract system, here we show that the Dna2 nuclease directly initiates the resection of clean DSBs by cleaving the 5′ strand DNA ∼10–20 nucleotides away from the ends. In the absence of Dna2, MRN together with CtIP mediate an alternative resection initiation pathway where the nuclease activity of MRN apparently directly cleaves the 5′ strand DNA at more distal sites. MRN also facilitates resection initiation by promoting the recruitment of Dna2 and CtIP to the DNA substrate. The ssDNA-binding protein RPA promotes both Dna2- and CtIP–MRN-dependent resection initiation, but a RPA mutant can distinguish between these pathways. Our results strongly suggest that resection of blocked and clean DSBs is initiated via distinct mechanisms.

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“…At single-ended breaks during replication, the nuclease activity of Mre11 was shown to be important for removal of Ku (6), suggesting that MRN catalytic processing of ends during S phase is important for recombination-mediated repair of single-ended breaks. While we and others have shown that MRN endonuclease activity is specifically stimulated by protein blocks on DNA ends (8)(9)(10)(11), we considered the possibility that DNA-PK itself constitutes a powerful block to resection, and that DNA-PK may create a critical barrier to end processing at single-ended breaks where there is no other DNA end to participate in end joining.…”

Section: Main Textmentioning

confidence: 99%

“…2A). Some of these phosphorylation events have been shown to promote DNA end resection in cells (8,9,(30)(31)(32)(33)(34)(35), most notably the CDK-dependent modification of T847. In addition, ATR phosphorylation of T859 also plays an important role in CtIP binding to chromatin and CtIPmediated resection (36).…”

Section: Main Textmentioning

confidence: 99%

“…1A, lanes 2 and 3). Formation of the cleavage product requires CtIP, which has been shown to promote the endonuclease activity of Mre11 on heterologous substrates (8,9). Mre11 nuclease activity in vitro is generally reported to be manganese-dependent; however, Mre11/Rad50 complexes from archaea and from T4 bacteriophage also exhibit nuclease activity in magnesium when physiologically relevant protein cofactors are present (13, 14).…”

Section: Introductionmentioning

confidence: 99%

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DNA-dependent protein kinase promotes DNA end processing by MRN and CtIP

Deshpande

Myler

Soniat

et al. 2018

Preprint

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Abstract:DNA-dependent Protein Kinase (DNA-PK) coordinates the repair of double-strand breaks through non-homologous end joining, the dominant repair pathway in mammalian cells. Breaks can also be resolved through homologous recombination during S/G 2 cell cycle phases, initiated by the Mre11-Rad50-Nbs1 (MRN) complex and CtIP-mediated resection of 5ʹ strands. The functions of DNA-PK are considered to be end-joining specific, but here we demonstrate that human DNA-PK also plays an important role in the processing of DNA double-strand breaks.Using ensemble biochemistry and single-molecule approaches, we show that the MRN complex in cooperation with CtIP is stimulated by DNA-PK to perform efficient endonucleolytic processing of DNA ends in physiological conditions. This activity requires both CDK and ATR phosphorylation of CtIP. These unexpected results could explain the absence of DNA-PK deletion mutations in the human population, as homologous recombination is an essential process in mammals.One sentence summary: An enzyme critical for non-homologous repair of DNA double-strand breaks also stimulates end processing for homologous recombination.. CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/395731 doi: bioRxiv preprint first posted online Aug. 19, 2018; 3 Main Text:DNA-dependent Protein Kinase consists of a catalytic kinase subunit (DNA-PKcs) and the DNA end-binding heterodimer of Ku70 and Ku80 (Ku). Together, these proteins form an end recognition complex (DNA-PK) that binds to DNA double-strand breaks within seconds of break formation (1). DNA-PK promotes the ligation of two DNA ends by ligase IV, aided by the accessory factors XLF, XRCC4, and APLF, and in some cases aided by limited end processing (1,2). The recognition and repair of DNA breaks by the end-joining machinery is generally considered to be the first and rapid phase of DNA repair, occurring within ~30 minutes of DNA damage, while homologous recombination is specific to the S and G 2 phases of the cell cycle and occurs over a longer time frame (3,4). DNA-PK is present at micromolar concentrations in cells (5) and binds DNA ends in all cell cycle phases, even during S phase at single-ended breaks (6). The MRN complex regulates the initiation of DNA end processing prior to homologous recombination by catalyzing the initial 5ʹ strand processing at blocked DNA ends and by recruiting long-range nucleases Exo1 and Dna2 (4, 7). At single-ended breaks during replication, the nuclease activity of Mre11 was shown to be important for removal of Ku (6), suggesting that MRN catalytic processing of ends during S phase is important for recombination-mediated repair of single-ended breaks. While we and others have shown that MRN endonuclease activity is specifically stimulated by protein blocks on DNA ends (8-11), we considered the possibility that DNA-P...

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Phosphorylated CtIP Functions as a Co-factor of the MRE11-RAD50-NBS1 Endonuclease in DNA End Resection

Cited by 242 publications

References 69 publications

Nonhomologous DNA end-joining for repair of DNA double-strand breaks

Nonhomologous DNA end-joining for repair of DNA double-strand breaks

Dna2 initiates resection at clean DNA double-strand breaks

DNA-dependent protein kinase promotes DNA end processing by MRN and CtIP

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