Paudyal SC et al. (2017), Dna2 initiates resection at clean DNA double-st...

XB-ART-54095

Nucleic Acids Res 2017 Nov 16;4520:11766-11781. doi: 10.1093/nar/gkx830.

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Dna2 initiates resection at clean DNA double-strand breaks.

Paudyal SC , Li S , Yan H , Hunter T , You Z .

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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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Species referenced: Xenopus laevis
Genes referenced: blm dna2 exo1 mre11 nbn nts pcna rbbp8 recql4 rpa1 wrn

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	Figure 1. Dna2 initiates the resection of clean DSB ends via cleavage of 5′ strand DNA. (A) A one-end 5′ 32P-labeled 2 kb DNA fragment (red star, 32P; purple circle, biotin) was incubated in NPE at room temperature. Reactions were terminated at the indicated times and resection products were resolved on a 16% polyacrylamide-urea gel. The top band represents the original DNA substrate that was ‘trapped’ in the loading wells of the gel. (B) Effects of Dna2 depletion on the resection of the radiolabeled DNA substrate depicted in (A) in the extract. (C) Purified recombinant Flag-Dna2(WT), Flag-Dna2(D278A) and Flag-Dna2(K655E) expressed in insect cells. (D) Comparison of the flap endonuclease activity of Flag-Dna2(WT), Flag-Dna2(K655E) and Flag-Dna2(D278A) towards a dsDNA substrate with a 5′ ssDNA flap in vitro. (E) Rescue of short 5′ endocleavage products in the Dna2-depleted extract by recombinant Flag-Dna2(WT) protein. (F) Comparison of the ability of recombinant Flag-Dna2(WT), Flag-Dna2(K655E) and Flag-Dna2(D278A) in restoring resection initiation in the Dna2-depleted extract.
	Figure 2. In the absence of Dna2, resection of clean DSBs is initiated through cleavage of 5′ strand DNA at more distal sites in a CtIP-dependent manner. (A) Effects of CtIP depletion on the initiation of resection of a 5′ radiolabeled clean DSB. (B) Effects of CtIP depletion on the generation of long endocleavage products in the Dna2-depleted extract. (C) Purified recombinant Flag-CtIP expressed in insect cells. (D) Rescue of long endocleavage products in the extract depleted of both Dna2 and CtIP by recombinant Flag-CtIP.
	Figure 3. The nuclease activity of MRN is important for the CtIP-dependent pathway, but apparently not for the Dna2-dependent pathway. (A) Effects of Mre11 depletion on the generation of short resection initiation products in the extract. (B) Effects of Mre11 depletion on the generation of long resection initiation products in the Dna2-depleted extract. (C) Effects of Mirin on the generation of short and long resection initiation products in the extract. (D) Comparison of the ability of recombinant MRN(WT) and M(H130N)RN to rescue short resection initiation products in the Mre11-depleted extract. (E) Comparison of the ability of recombinant MRN(WT) and M(H130N)RN to rescue long resection initiation products in the extract depleted of both Dna2 and Mre11.
	Figure 4. MRN promotes the recruitment of Dna2 and CtIP to DNA substrate to initiate resection. (A) Effects of NBS1 depletion on the generation of short resection initiation products in the extract. (B) Effects of NBS1 inhibitory antibodies on the generation of short resection initiation products in the extract. (C) Effects of NBS1 inhibitory antibodies on the generation of long resection initiation products in the Dna2-depleted extract. (D) Effects of NBS1 inhibitory antibodies on the binding of NBS1, CtIP, Dna2, Exo1, RPA and PCNA to the DNA substrate in the extract. (E) Effects of NBS1 inhibitory antibodies on the binding of NBS1, CtIP, Exo1, RPA and PCNA to the DNA substrate in the Dna2-depleted extract.
	Figure 5. RPA plays a key role in resection initiation by both the Dna2- and CtIP-dependent pathways. (A) Effects of RPA depletion on the generation of short resection initiation products in the extract. (B) Effects of RPA depletion on the generation of long resection initiation products in the Dna2-depleted extract. (C) Purified recombinant Flag-Dna2(WT) and Flag-Dna2(27–1053) expressed in insect cells. (D) Comparison of the flap endonuclease activity of Flag-Dna2(WT) and Flag-Dna2(27–1053) in vitro. (E) Co-immunoprecipitation of RPA with Flag-Dna2(WT) and Flag-Dna2(27–1053) added to the extract. (F) Comparison of the ability of Flag-Dna2(WT) and Flag-Dna2(27–1053) to rescue short resection initiation products in the Dna2-depleted extract. (G) Effects of RPA depletion on the binding of NBS1, CtIP, Dna2, Exo1 and PCNA to the DNA substrate in the extract. (H) Effects of RPA depletion on the binding of NBS1, CtIP, Exo1 and PCNA to the DNA substrate in the Dna2-depleted extract. (I) Comparison of the ability of RPA(WT) and RPA(1NΔ) to rescue short resection initiation products in the RPA-depleted extract. (J) Comparison of the ability of RPA(WT) and RPA(1NΔ) to rescue long resection initiation products in the extract depleted of both Dna2 and RPA.
	Figure 6. A model for resection initiation at clean or blocked DSBs. DNA end resection at a blocked DSB with protein or chemical adducts (denoted by pink rectangle) is initiated through endocleavage by the MRN nuclease in conjunction with CtIP, with the nuclease activity of MRN being responsible for the cleavage. By contrast, resection initiation at a clean DSB with free DNA ends is initiated by Dna2, which cleaves the 5′ strand DNA ∼10–20 nts away from the end. The MRN complex promotes this step by facilitating the recruitment of Dna2 to the DNA end. In the absence of Dna2, CtIP–MRN mediate an alternative pathway of resection initiation, with the endonuclease activity of MRN cleaving the 5′ strand DNA ≥45 nts away from the end. Both Dna2 and CtIP–MRN pathways of resection initiation apparently require DNA helicases such as WRN, BLM, RECQL4 and likely Dna2, which unwind DNA ends to generate ssDNA for cleavages. The ssDNA-binding protein RPA binds the resulting ssDNA and promotes resection initiation by both pathways. Dna2-mediated end cleavage requires the direct interaction between Dna2 and the N-terminus of RPA1 in the RPA complex, but this domain of RPA1 is dispensable for the CtIP–MRN pathway of resection initiation.

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