Fig 2: SMC1A K579 is acetylated by CBP and deacetylated by SIRT2.(A) Flag-tagged CBP, P300, p300/CBP-associated factor (PCAF), or Myc-tagged GCN5 was individually cotransfected with Flag-SMC1A into HEK293T cells. SMC1A acetylation was detected by IP using Ac-K antibody. (B) The interaction between endogenous SMC1A and CBP was detected by IP–Western blot. (C) Exogenously expressed SMC1A interacts with endogenous CBP. Flag-tagged SMC1A plasmid was transfected into HEK293T cells. (D) Sequence alignment of SMC1A homologs surrounding K579 in various species. The bold red indicates the acetylated lysine residues at 579 of SMC1A. (E) Acetylation of SMC1A WT, K579R, and K579Q. Constructs were transfected into HEK293T cells, and whole lysates were immunoprecipitated, followed by Western blot to detect acetylation. (F) Flag-tagged SMC1A plasmid was transfected into HEK293T cells. Immunoprecipitated Flag-SMC1A was probed with the site-specific K579 acetylation antibody (K579Ac). (G) HCT116 cells expressing Flag-tagged SMC1A were cotransfected with Myc-tagged SIRT2 with or without the SIRT2-specific inhibitor AGK2 (10 µM, 24 hours). SMC1A acetylation was detected by IP using the K579Ac antibody. (H) Myc-tagged SIRT2 was transfected into HCT116 cells. Endogenous SMC1A acetylation was detected using the K579Ac antibody.

Fig 3: K579 acetylation of SMC1A inhibits tumor growth.(A) Mice bearing HCT116 xenograft (SMC1A WT or K579R) were treated by direct intratumoral injection with either the control solvent (50 µl of DPH solution) or AGK2 (100 µM in 50 µl of DPH solution) three times per week. The volumes of the xenograft tumors in nude mice are shown after the 20-day treatment (n = 8). Data were expressed as means ± SEM. ns, no difference; *P < 0.05; **P < 0.01; ***P < 0.001. (B and C) Xenografts were performed using the HCT116-shSMC1A cells reexpressing SMC1A WT, K579Q, or K579Q-DD mutant. Gross images, volume curves (B), and weight (day 26) (C) of xenograft tumors in nude mice (n = 10). Data were expressed as means ± SEM. **P < 0.01; ***P < 0.001. (D) Cells overexpressing myc-SIRT2 were treated with 5-FU (2.5 µg/ml) on different days. Cell viability was measured by the CCK8 assay. Data are means ± SEM. *P < 0.05; ***P < 0.001. (E and F) Myc-tagged SIRT2 was transfected into HCT116 cells treated with 5-FU (2.5 µg/ml, 48 hours). PCNA (E), C-PARP, and the cleaved caspase-3 (C-Cas3) (F) were detected by Western blot. (G) Cells overexpressing myc-SIRT2 were treated with 2 µM oxaliplatin on different days. Cell viability was measured by the CCK8 assay. Data are means ± SEM. ***P < 0.001. (H and I) Myc-tagged SIRT2 was transfected into HCT116 cells treated with oxaliplatin (2 µM, 48 hours). PCNA (H), C-PARP, and the cleaved caspase-3 (C-Cas3) (I) were detected by Western blot. (J to M) Cells were treated with oxaliplatin (J) and 5-FU (L) at different concentrations for 48 hours. Cell viability was measured by the CCK8 assay. Data were expressed as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Cells were treated with oxaliplatin (2 µM) (K) and 5-FU (2.5 µg/ml) (M) for 48 hours. The levels of C-PARP and cleaved caspase-3 (C-Cas3) were detected by Western blot.

Fig 4: ATM and ATM kinase activity/signalling in cells from ataxia telangiectasia patients 1–6. Assay showing the presence or absence of ATM in cells from each patient and whether this was associated with some activity/signalling ability. Cells were either irradiated (IR) with 2-Gy x-rays or not irradiated. The left-hand section show the results of the three classical patients, the middle section the results of variant patient 2, and the right-hand section the results of variants 4 and 5. Left-hand section, lanes 1 & 2—positive control. (A) Cells from a normal individual showing presence of ATM protein (B) and normal ATM activity/signalling showing phosphorylation of the targets SMC1 ser966 (C), KAP-1 ser 824 (E), Nbn ser343 (G), and CREB ser121 (I). There is a strong signal for each of these in lane 2 after activation. The total levels of SMC1, KAP-1, Nbn, and CREB are also shown in (D, F, H, J), respectively. Lanes 3 and 4—negative control. Cell lysate from a classical A-T patient with mutations ATM, c.1355delC; p.(Thr452AsnfsTer21) and c.3802delG; p.(Val1268Ter). Both are frameshift mutations leading to instability and loss of the ATM from both alleles. There is no differential phosphorylation of these targets in this patient’s cells inane 4—consistent with the patient having A-T; there is no ATM (B). Lanes 5 and 6 show cell lysates from patient 6, lanes 7 and 8 lysates from patient 3, and lanes 9 and 10 lysates from patient 1. Just as with the negative control (lane 4), lysates from these three patients show absence of ATM protein (B) and absence of a differentially increased phosphorylation induced by irradiation, as indicated by absence of phosphorylation of SMC1 ser966, KAP-1 ser824, Nbn ser343, and CREB ser121 in lanes 6, 8, and 10, respectively. Middle section: lanes 11 and 12 show lysates from the same normal control as in lanes 1 and 2. Lanes 13 and 14 are cell lysates from the same classical A-T patient as in lanes 3 and 4. Lanes 15 and 16 are lysates from variant patient 2. (B) confirms that the lysate shows a low level of ATM protein. In contrast with the negative control (lane 14), this lysate shows a clear low level of ATM kinase activity/signalling as indicated by the presence of some moderate phosphorylation of SMC1 ser966, KAP-1 ser824, Nbn ser343, or CREB ser121 in lane 16. Right-hand section, lanes 17 and 18 and 19 and 20 are lysates from variant patients 4 and 5. The positive and negative controls have been removed but were the same as in lanes 1–4 and 11–14. (B) confirms that the variant lysates show a low level of ATM protein. In contrast to the negative controls in lanes 4 and 14, these lysates also show a clear low level of ATM that has some retained kinase activity/signalling as indicated by the presence of moderate phosphorylation of SMC1 ser966, KAP-1 ser824, and Nbn ser343 in particular.

Fig 5: Normal ATM signaling in patient cells.ATM from the lymphoblastoid cell line of II-5 is able to autophosphorylate (panel A), shows a normal level (panel B, lanes 5 & 6) as well as normal signalling as indicated by phosphorylation of ATM targets Smc1Ser966 (panel C) and NbnSer343 (panel E), as is the case for the normal control (lanes 1 & 2). Lanes 3 & 4 show the A-T control, where there is no ATM (panel B) and no evidence of ATM signalling. Panels D & F show the total levels of Smc1 and Nbn respectively.