Hashimoto Y and Tanaka H (2022), Mre11 exonuclease activity promotes irreversibl...

XB-ART-58934

Life Sci Alliance 2022 Mar 15;56:. doi: 10.26508/lsa.202101249.

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Mre11 exonuclease activity promotes irreversible mitotic progression under replication stress.

Hashimoto Y , Tanaka H .

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Mre11 is a versatile exo-/endonuclease involved in multiple aspects of DNA replication and repair, such as DSB end processing and checkpoint activation. We previously demonstrated that forced mitotic entry drives replisome disassembly at stalled replication forks in Xenopus egg extracts. Here, we examined the effects of various chemical inhibitors using this system and discovered a novel role of Mre11 exonuclease activity in promoting mitotic entry under replication stress. Mre11 activity was necessary for the initial progression of mitotic entry in the presence of stalled forks but unnecessary in the absence of stalled forks or after mitotic entry. In the absence of Mre11 activity, mitotic CDK was inactivated by Wee1/Myt1-dependent phosphorylation, causing mitotic exit. An inhibitor of Wee1/Myt1 or a nonphosphorylatable CDK1 mutant was able to partially bypass the requirement of Mre11 for mitotic entry. These results suggest that Mre11 exonuclease activity facilitates the processing of stalled replication forks upon mitotic entry, which attenuates the inhibitory pathways of mitotic CDK activation, leading to irreversible mitotic progression and replisome disassembly.

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Species referenced: Xenopus laevis
Genes referenced: cdk1 mre11

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	Figure 1. Mre11 exonuclease activity is required for mitotic entry and mitotic replisome disassembly in the presence of stalled replication forks.(A) The basic experimental design. In the first reaction, sperm nuclei (5,000/μl) were incubated for 60 min in 20 μl of S-phase/interphase egg extract (S-extract) with 10 μg/ml of aphidicolin. In the second reaction, 20 μl of M-phase egg extract (M-extract) was added to the first reaction mixture together with an additional 10 μg/ml of aphidicolin to induce mitotic entry and replisome disassembly. After 60-min incubation of the second reaction, chromatin fractions were isolated and analyzed by immunoblotting. (B, D) The effects of inhibitors on mitotic entry and replisome disassembly. The second reaction was performed in the absence or presence of 100 μg/ml of His-p27 (27), 50 μM mirin (Mi), 100 μM mirin (Mi), 100 μM NMS-873 (N), 10 μM MLN-4924 (ML), 50 μM PFM01 (P), or 100 μM PFM01 (P**). (C) Role of phosphorylation in mitotic mobility shifts of Mre11 and Cut5/TopBP1. The isolated chromatin fractions were treated with or without calf intestine alkaline phosphatase (+/−CIAP) and analyzed by immunoblotting. In (B) and (C), 0.5 μl of S-phase and M-phase extracts (ext S, M) was analyzed by immunoblotting as controls. (E) Comparison of mitotic entry between replication-complete and replication-incomplete nuclei. The first reaction was performed for 90 min with (replication-incomplete) and without (replication-complete) aphidicolin.Source data are available for this figure.
	Figure 2. Mre11 facilitates the processing of stalled forks during the early stages of mitotic entry.(A) The requirement of Mre11 activity for replisome disassembly during replication termination in the S-phase. Sperm nuclei (5,000/μl) were incubated for the indicated times in 20 μl of S-extract in the absence or presence of 100 μM mirin or 100 μM NMS-873. The chromatin fractions were isolated and analyzed by immunoblotting. (B) The requirement of Mre11 activity for mitotic progression after mitotic entry. A similar experiment to that shown in Fig 1B was performed under the following conditions: 100 μM mirin was added together with M-extract (+Mi(0′)) or at 15 (+Mi(15′)) and 30 min (+Mi(30′)) after the addition of M-extract. After 20, 40, or 60 min for the second reaction, the chromatin fractions were isolated and analyzed by immunoblotting. (C) The requirement of interphase Mre11 activity for mitotic entry. In the first reaction, sperm nuclei (5,000/μl) were incubated for 60 min in 20 μl of S-extract in the absence or presence of 100 μM mirin. In the second reaction, 30 μl of S-extract and 50 μl of M-extract were added (to dilute sperm nuclei to 1,000/μl) with or without additional mirin to adjust the final concentrations of mirin to 0, 20, and 100 μM as indicated. The concentrations of aphidicolin were kept at 10 μg/ml throughout the experiment in all the cases. His-p27 was added as controls. After 60-min incubation, the chromatin fractions were isolated and analyzed by immunoblotting. (D) The requirement of mitotic CDK activity for mitotic progression after mitotic entry. (B) The same experiment as shown in (B) was performed using His-p27 instead of mirin, and the chromatin fractions were isolated and analyzed by immunoblotting. In (B) and (D), 0.5 μl of S-phase and M-phase extracts (ext S and M) was also analyzed by immunoblotting as controls.Source data are available for this figure.
	Figure 3. Mitotic CDK activity is inactivated by Wee1/My1 in the absence of Mre11-dependent processing of stalled forks.(A) Inactivation of mitotic CDK activity in the presence of mirin. The same experiment with that shown in Fig 1B was performed with the indicated conditions. After 0-, 15-, 30-, and 60-min incubation of the second reaction, 1 μl of the whole extract was sampled and analyzed by immunoblotting. (B) The dependence of CDK inactivation on Wee1/Myt1 kinases. The same experiment with that shown in Fig 1B was performed with the indicated conditions. After 0-, 10-, and 20-min incubation of the second reaction, 1 μl of the whole extract was sampled and analyzed by immunoblotting (lanes 1–15). As controls, S-phase and M-phase extracts (S-ext and M-ext) were incubated without nuclei for 40 min in the absence and presence of mirin, and 1 μl of each were sampled and analyzed by immunoblotting. (C) The recovery of mitotic entry by the inhibition of Wee1/Myt1. The same experiment with that shown in Fig 1B was performed with the indicated conditions, and the chromatin fractions were isolated and analyzed by immunoblotting. (A, B, C, D) His-p27 (p27), mirin, and PD166285 (PD) were used at final concentrations of 100 μg/ml, 100 μM, and 10 μM, respectively. (D, E) The effect of mirin for the ATR-Chk1 checkpoint activity. The same experiment with that shown in Fig 1B was performed with the indicated conditions, and the nuclear fractions were isolated and analyzed by immunoblotting. The relative mean values of Chk1-P intensities of three independent experiments were shown in the graph using Cut5/TopBP1 intensities as loading controls. Error bar, ± S.D. P-values were calculated by the unpaired t test (one-tailed). ns, not significant.Source data are available for this figure.
	Figure 4. A nonphosphorylatable CDK1 mutant can promote mitotic entry without Mre11 exonuclease activity.(A) The ability of the recombinant CDK complexes of MBP–cyclin B1-ΔN90 with GST-CDK1 wild type (CDK-WT, WT) or GST-CDK1-T14A/Y15F (CDK-AF, AF) to promote mitotic entry in the absence or presence of replication stress. In the first reaction, sperm nuclei (5,000/μl) were incubated for 80 min in 20 μl of S-phase egg extract with (+Aph) or without (−Aph) 10 μg/ml of aphidicolin. In the second reaction, CDK-WT or CDK-AF was added at 2.2 μM and incubated for the indicated times (5, 20, 40, and 60 min). 1 μl of the whole extracts were sampled and analyzed by immunoblotting. The positions of recombinant GST-CDK1 (rec) and endogenous CDK1 (endo) are indicated by filled and open triangles, respectively. (B, C) Restoration of mitotic entry by the nonphosphorylatable CDK1 mutant. (A) A similar experiment with that shown in (A) was performed with the indicated conditions. PD166285 (PD) and mirin were added at 5 and 100 μM, respectively. (B, C) The whole extract (B) and the chromatin fractions (C) were analyzed by immunoblotting.Source data are available for this figure.
	Figure 5. A model for mitotic entry in the presence of stalled forks.Active pathways are indicated with bold font/lines and large arrows, whereas inactive pathways are indicated with regular font/dotted lines and small arrows.

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Ayoub, The carboxyl terminus of Brca2 links the disassembly of Rad51 complexes to mitotic entry. 2009, Pubmed