铅和硒对端粒长度、端粒酶及端粒结合蛋白的影响

以酿酒酵母细胞为实验材料 ,在分子水平上研究铅 (Pb)和硒 (Se)对端粒长度、端粒酶及端粒结合蛋白的影响。结果发现 :与对照组相比 ,添加 1mg/LPb的培养基中培养 10 0代后的酿酒酵母细胞中端粒DNA的平均长度缩短 ,端粒结合蛋白Rap1p含量减少 ,而且Rap1p蛋白的二级结构受到扰动、端粒酶活性降低、43%的细胞死亡。加 1mg/LSe培养 10 0代后的酿酒酵母细胞与对照组相比 ,细胞中端粒平均长度增加 ,Rap1p蛋白浓度和二级结构保持稳定 ,端粒酶活性增加 ,细胞正常存活。以上结果表明 ,Pb对酿酒酵母细胞端粒有损伤 ,而且其损伤在子代细胞中有累积效应 ;而Se对Pb损伤具有一定程度的修复保护作用 ,适量给机体补Se对抑制细胞损伤和衰老有一定作用。由于端粒的特殊结构特征 ,推断Pb和Se是通过作用于端粒酶及端粒结合蛋白而间接影响端粒长度的     

基因组编辑对酿酒酵母DNA的损伤作用及修复响应

【目的】为了研究基因组编辑工具CRISPR/Cas9和CRISPR/Cpf1所产生的DNA双链断裂(DNA doublestrandbreak,DSB)对酿酒酵母DNA的损伤作用及修复响应情况,对比化学物质甲基磺酸甲酯(methyl methanesulfonate,MMS)对酿酒酵母基因组DNA的损伤和修复,阐明编辑细胞在细胞水平和转录水平上的变化。【方法】起始细胞分为两种情况,包括未进行细胞周期同步化和被α-因子同步化细胞周期至G0/G1期。检测CRISPR/Cas9和CRISPR/Cpf1处理后编辑细胞的生长情况。利用流式细胞术检测编辑细胞的细胞周期延滞的情况。利用荧光定量PCR检测编辑细胞和MMS处理细胞后DNA损伤响应关键基因转录表达水平的变化情况。【结果】起始细胞无论是未同步化还是同步化,其生长均受到基因组编辑抑制,细胞存活率降低,细胞周期被滞留在G2/M期,而MMS处理导致细胞周期S期的滞留。此外,随编辑时间的延长,突变率增加,细胞存活率降低。CRISPR/Cpf1编辑细胞的突变率和存活率均低于CRISPR/Cas9,由此可见,CRISPR/Cpf1对细胞的损伤强度高于CRISPR/Cas9。两种编辑均诱导酵母DNA损伤响应关键基因RNR3及HUG1转录水平显著上调,并且CRISPR/Cpf1介导的上调幅度大于CRISPR/Cas9,但两者均低于MMS的处理。【结论】本研究解析了CRISPR/Cas9和CRISPR/Cpf1介导的基因组编辑在细胞水平和转录水平上对DNA损伤作用及修复响应,初步揭示了酿酒酵母应对不同类型的DSB损伤时响应程度的差异,为提高基因组编辑工具的编辑能力和评估基因编辑安全性提供了重要依据。     

WEB2基因在酿酒酵母S期检查点通路上调控RNR3基因表达

WEB2基因参与酿酒酵母S期检查点调控机制,而RNR3基因位于该调控通路末端,DNA损伤或合成阻断时,S期检查点通路诱导RNR3过度表达。因此,通过确定WEB2在该检查点通路上是否参与调控RNR3基因的表达,将有助于进一步明确WEB2基因在检查点通路上的工作位点,了解WEB2基因如何发挥检查点调控功能。构建RNR3-LacZ基因融合质粒,用于检测酵母细胞内RNR3基因的诱导性。诱导性可以通过测定β-半乳糖苷酶的活性而得知。利用DNA损伤药物甲磺酸甲酯(MMS)及DNA合成阻断剂羟基脲(HU)处理酵母细胞,测定WEB2基因突变株和野生株细胞内RNR3基因的诱导性。结果,WEB2突变株细胞中诱导活性分别增加(8.27±0.38)倍和(9.55±0.24)倍,而野生株分别增加了(83.32±2.42)倍和(124.67±2.87)倍。反映RNR3基因在WEB2突变株中的诱导性低于野生株。同RAD53突变株相比,后者的RNR3基因的诱导性更低,仅为(2.37±0.18)倍和(2.91±0.13)倍。说明WEB2基因突变影响S期检查点通路的信号传递至RNR3基因,所以在酿酒酵母S期检查点通路上,WEB2工作在RNR3基因上游,参与调控RNR3的表达,但调控能力不如RAD53基因强。     

大黄素对宫颈癌Hela细胞端粒损伤作用的研究

该文探究了抗肿瘤药物、化疗增敏剂大黄素对人宫颈癌Hela细胞端粒和端粒酶活性的影响。利用磷酸化组蛋白H2AX(γ-H2AX)免疫荧光–端粒荧光原位杂交技术检测端粒区特异性DNA损伤水平。利用中期染色体–端粒荧光原位杂交技术检测端粒异常信号,包括多端粒信号(multitelomeric signals,MTSs)和端粒信号缺失(signal free ends,SFEs)。利用荧光定量PCR方法和端粒重复扩增程序分别检测端粒相对长度和端粒酶活性。结果显示,与0μmol/L对照组相比,20μmol/L大黄素处理Hela细胞48 h能够诱导端粒长度缩短至80%,同时还发现端粒功能障碍损伤灶(telomere dysfunction induced foci,TIFs)和端粒异常信号增多,包括MTSs由1.65%增加至3.98%(P0.01)、SFEs由2.74%增加至8.49%(P0.01)。同时,结果还发现,大黄素处理后,Hela细胞端粒酶活性显著升高,10μmol/L和20μmol/L大黄素处理48 h后,端粒酶活性分别升高为对照组的1.42倍(P0.05)和1.92倍(P0.01)。综上,实验结果表明,大黄素的急性暴露能够引起端粒功能障碍以及端粒酶活性升高,后者可能与端粒损伤后修复有关。     

化疗药物对人胃癌SGC-7901细胞系端粒酶活性影响

目的　研究常见化疗药物对人胃癌SGC 790 1细胞系端粒酶活性影响。方法　MTT (噻唑蓝 )法测定化疗药物顺铂、丝裂霉素、阿霉素、 5 氟尿嘧啶、氟铁龙 (脱氧氟尿苷 )对人胃癌SGC 790 1细胞系毒性作用的半数抑制(IC5 0 )浓度 ;用TRAP ELISA法测定 4 0×IC5 0浓度化疗药物作用于SGC 790 1细胞 4h、 2 8h端粒酶活性及IC5 0浓度IC5 0化疗药物作用于SGC 790 1细胞 2 4h、 72h、 12 0h细胞端粒酶活性。结果　高浓度 ,低浓度顺铂、丝裂霉素对SGC 790 1细胞端粒酶活性有完全抑制作用 ;而阿霉素、 5 氟尿嘧啶、氟铁龙对SGC 790 1细胞端粒酶活性有部分抑制作用。结论　阿霉素、 5 氟尿嘧啶、氟铁龙对SGC 790 1细胞端粒酶活性有轻度抑制作用 ;顺铂、丝裂霉素能完全抑制端粒酶活性 ,呈时间浓度效应     

细胞周期及DNA损伤剂引起的细胞NAD含量变化及其意义

本文观察了FL细胞中ADP-核糖基转移酶(ADPRT)底物NAD含量的细胞周期性变化及其与DNA复制之间的关系。FL细胞NAD含最在G_1期最高,而在S期DNA合成高峰后0—3小时(S/G_2期)达到最低点。ADPRT抑制剂3 AB能够抑制NAD含量的细胞周期性变化,而且S期DNA合成亦受到抑制,并呈现S期延长,提示ADP-核糖基化作用可能参与DNA复制过程。本文还观察了三种DNA损伤剂MNNG、MMS及4NQO对处于细胞周期不同时相的FL细胞NAD含量的影响,以及ADPRT抑制剂3 AB及尼克酰胺对此影响的作用。证明ADPRT抑制剂可以特异地抑制DNA损伤性NAD含量下降而对正常FL细胞NAD含量及代谢抑制剂2,4-DNP所致的NAD含量下降没有影响。从而有可能建立一个以测量细胞内NAD含量为指标的简便、快速、特异的检测DNA损伤因子的方法。     

端粒酶抑制剂对不同非整倍体状态的人结肠癌HCT116细胞增殖及凋亡的影响

该文研究端粒酶在人结肠癌HCT116高非整倍体变异组及低非整倍体变异组细胞中的表达差异及其端粒酶抑制剂3′-叠氮-3′-脱氧胸苷(3′-Azido-3′-deoxythymidine,AZT)对2组细胞增殖及凋亡的影响。取HCT116细胞加入盐酸强力霉素16 h后撤药,称为高非整倍体变异组;另取HCT116细胞不作任何处理,设为对照组,称为低非整倍体变异组;在撤药后第11 d,采用100、250μmol/L的AZT处理2组细胞72 h,并分别设立空白对照组(未加AZT的高非整倍体变异组及低非整倍体变异组)。采用染色体滴定法进行染色体计数。采用Western blot检测MAD2L1、PUMA、BAX、P21、γ-H2AX蛋白质水平。采用荧光定量PCR检测h TERT、PUMA、BAX、NOXA、P21基因表达,端粒酶活性试剂盒检测端粒酶活性,CCK-8法检测细胞生存率。实验结果表明,采用盐酸强力霉素诱导HCT116细胞的非整倍体率可达到77.33%;高非整倍体变异组细胞h TERT基因的表达及端粒酶活性明显高于低非整倍体变异组;加入AZT后,高非整倍体变异组P21、γ-H2AX蛋白质水平上升程度较整倍体明显,低非整倍体变异组PUMA、BAX蛋白质水平上升程度较高非整倍体变异组明显。该研究表明,盐酸强力霉素可以诱导非整倍体形成,高非整倍体变异组的端粒酶活性及h TERT基因表达高于低非整倍体变异组,AZT可以对高非整倍体变异组和低非整倍体变异组细胞产生增殖抑制作用、DNA损伤作用、细胞周期阻滞作用、诱导凋亡作用。     

盐胁迫下沙冬青细胞端粒酶活性的变化与DNA稳定性的关系

沙冬青(Ammopiptanthus mongolicus)是亚洲中部荒漠地区的常绿阔叶灌木,是古老的第三纪孑遗植物,具有极强的抗逆能力。旨在探究盐胁迫下沙冬青细胞端粒酶活性变化与染色体DNA稳定性的关系。结果表明,在100 mmol/L NaCl的低浓度盐胁迫处理下,随着处理时间的增加,端粒酶活性没有增加,未出现明显的DNA降解现象;当在500 mmol/L高浓度盐胁迫时,处理的初期端粒酶活性迅速的增加,但随着处理时间的延长,端粒酶活性下降,此时DNA未明显降解;当移除NaCl胁迫,端粒酶活性增加1.4倍,DNA保持稳定。因此推测,在盐胁迫下,沙冬青细胞端粒酶活性的维持对避免细胞遭受不可逆损伤,保持染色体DNA稳定性具有一定的作用。     

盐胁迫下胡杨和合作杨悬浮细胞端粒酶与抵御氧化损伤的关系

胡杨是我国西北干旱盐碱荒漠和戈壁地带唯一能形成森林的高大乔木树种,具有较高抗盐性。旨在探究盐胁迫下细胞端粒酶活性变化与氧化损伤的关系,以抗盐的胡杨和盐敏感的合作杨悬浮细胞为材料,检测了Na Cl盐胁迫条件下胡杨和合作杨细胞生长、超氧阴离子自由基与丙二醛含量以及端粒酶活性的变化。结果表明,在正常生长条件下,胡杨和合作杨细胞符合S型生长曲线,且合作杨细胞的生长量高于胡杨细胞;在盐胁迫100 mmol/L时胡杨在培养7 d内细胞活力高于对照(0 mmol/L),即使盐浓度达到300 mmol/L时15 d胡杨细胞依然保持一定的细胞活力,而较低盐处理对合作杨细胞活力影响即较大,当盐浓度达到300 mmol/L时合作杨在第5 d细胞活力已接近零;与合作杨相比,低浓度盐胁迫(100 mmol/L)使胡杨细胞内超氧阴离子自由基含量增加,端粒酶活性较高,而合作杨丙二醛含量显著增加;低浓度(20 mmol/L)H2O2诱导了胡杨细胞端粒酶活性,合作杨的变化则不显著,但高浓度(200 mmol/L)Na Cl和高浓度(50 mmol/L)H_2O_2可能造成了胡杨和合作杨氧化损伤,端粒酶活性降低。抗盐性强的胡杨细胞端粒酶对抵御细胞内氧化损伤具有一定作用。     

渥曼青霉素对端粒DNA链断裂重连接作用的影响

端粒是位于真核细胞染色体末端的DNA-蛋白质复合体,在维持染色体稳定上起着重要的作用,并且与细胞的衰老、癌变有着密切的关系。本实验观察了DNA依赖性蛋白激酶(DNA-dependent protein kinase,DNA-PK)抑制剂-渥曼青霉素(wortmannin,WM)对H2O2诱导的HeLa细胞端粒DNA链断裂重连接效应。结果表明WM能够显著地抑制H2O2诱导的HeLa细胞端粒DNA链断裂后的重连接作用,提示DNA-PK参与了端粒DNA链断裂损伤的修复过程。     

Studying the synergistic damage effects induced by 1.8 GHz radiofrequency field radiation (RFR) with four chemical mutagens on human lymphocyte DNA using comet assay in vitro

The aim of this investigation was to study the synergistic DNA damage effects in human lymphocytes induced by 1.8 GHz radiofrequency field radiation (RFR, SAR of 3 W/kg) with four chemical mutagens, i.e. mitomycin C (MMC, DNA crosslinker), bleomycin (BLM, radiomimetic agent), methyl methanesulfonate (MMS, alkylating agent), and 4-nitroquinoline-1-oxide (4NQO, UV-mimetic agent). The DNA damage of lymphocytes exposed to RFR and/or with chemical mutagens was detected at two incubation time (0 or 21 h) after treatment with comet assay in vitro. Three combinative exposure ways were used. Cells were exposed to RFR and chemical mutagens for 2 and 3h, respectively. Tail length (TL) and tail moment (TM) were utilized as DNA damage indexes. The results showed no difference of DNA damage indexes between RFR group and control group at 0 and 21 h incubation after exposure (P>0.05). There were significant difference of DNA damage indexes between MMC group and RFR+MMC co-exposure group at 0 and 21 h incubation after treatment (P<0.01). Also the significant difference of DNA damage indexes between 4NQO group and RFR+4NQO co-exposure group at 0 and 21 h incubation after treatment was observed (P<0.05 or P<0.01). The DNA damage in RFR+BLM co-exposure groups and RFR+MMS co-exposure groups was not significantly increased, as compared with corresponding BLM and MMS groups (P>0.05). The experimental results indicated 1.8 GHz RFR (SAR, 3 W/kg) for 2h did not induce the human lymphocyte DNA damage effects in vitro, but could enhance the human lymphocyte DNA damage effects induced by MMC and 4NQO. The synergistic DNA damage effects of 1.8 GHz RFR with BLM or MMS were not obvious.     

The G2/M DNA damage checkpoint inhibits mitosis through Tyr15 phosphorylation of p34cdc2 in Aspergillus nidulans.

It is possible to cause G2 arrest in Aspergillus nidulans by inactivating either p34cdc2 or NIMA. We therefore investigated the negative control of these two mitosis-promoting kinases after DNA damage. DNA damage caused rapid Tyr15 phosphorylation of p34cdc2 and transient cell cycle arrest but had little effect on the activity of NIMA. Dividing cells deficient in Tyr15 phosphorylation of p34cdc2 were sensitive to both MMS and UV irradiation and entered lethal premature mitosis with damaged DNA. However, non-dividing quiescent conidiospores of the Tyr15 mutant strain were not sensitive to DNA damage. The UV and MMS sensitivity of cells unable to tyrosine phosphorylate p34cdc2 is therefore caused by defects in DNA damage checkpoint regulation over mitosis. Both the nimA5 and nimT23 temperature-sensitive mutations cause an arrest in G2 at 42 degrees C. Addition of MMS to nimT23 G2-arrested cells caused a marked delay in their entry into mitosis upon downshift to 32 degrees C and this delay was correlated with a long delay in the dephosphorylation and activation of p34cdc2. Addition of MMS to nimA5 G2-arrested cells caused inactivation of the H1 kinase activity of p34cdc2 due to an increase in its Tyr15 phosphorylation level and delayed entry into mitosis upon return to 32 degrees C. However, if Tyr15 phosphorylation of p34cdc2 was prevented then its H1 kinase activity was not inactivated upon MMS addition to nimA5 G2-arrested cells and they rapidly progressed into a lethal mitosis upon release to 32 degrees C. Thus, Tyr15 phosphorylation of p34cdc2 in G2 arrests initiation of mitosis after DNA damage in A. nidulans.     

The combined action of chemical carcinogens on DNA repair in human cells

Summary Excision repair was studied in normal human and ataxia telangiectasia (AT) cells proficient in repair of UV and its mimetic chemicals, and in xeroderma pigmentosum group C (XP C) cells (deficient in repair of UV and its mimetics), after treatment with several combinations of chemical carcinogens, by the photolysis of bromodeoxyuridine incorporated into parental DNA during repair. Results indicate that repair was additive in AT, and XP C cells treated with N-acetoxy-2-acetylaminofluorene (AAAF) plus ethyl methanesulfonate (EMS) or methyl methanesulfonate (MMS) indicating that there are different rate limiting steps for removal of both types of damage. Data on the combinations of 4-nitroquinoline 1-oxide (4NQO) plus MMS or EMS are difficult to interpret, but they do not indicate inhibition of DNA repair.Research carried out under the auspices of the U.S. Dept. of Energy     

The MMS22L-TONSL complex mediates recovery from replication stress and homologous recombination

Genome integrity is jeopardized each time DNA replication forks stall or collapse. Here we report the identification of a complex composed of MMS22L (C6ORF167) and TONSL (NFKBIL2) that participates in the recovery from replication stress. MMS22L and TONSL are homologous to yeast Mms22 and plant Tonsoku/Brushy1, respectively. MMS22L-TONSL accumulates at regions of ssDNA associated with distressed replication forks or at processed DNA breaks, and its depletion results in high levels of endogenous DNA double-strand breaks caused by an inability to complete DNA synthesis after replication fork collapse. Moreover, cells depleted of MMS22L are highly sensitive to camptothecin,?a topoisomerase I poison that impairs DNA replication progression. Finally, MMS22L and TONSL are necessary for the efficient formation of RAD51 foci after DNA damage, and their depletion impairs homologous recombination. These results indicate that MMS22L and TONSL are genome caretakers that stimulate the recombination-dependent repair of stalled or collapsed replication forks.     

A technique for the measurement of breakage and repair of DNA alkylated in vivo.

The alkaline elution technique has been adapted for use in the assessment of DNA damage induced in the livers and lungs of mice after administration of an alkylating agent, methylemthanesulfonat (MMS). At 4 h after administration of MMS, damage ot DNA was readily demonstrable; the damage was repaired in liver by 24 h. The lung, particularly of the A/J mouse, exhibited an increased alkaline elution rate when compared to C57BL/6J, and repair was not entirely complete (as judged from the rate of alkaline elution of DNA) by 24 h. The rate of elution was dependent upon temperature. It is believed that this adaptation should have great utility in examining DNA repair in vivo.     

Characterization of MMS-sensitive mutants of Neurospora crassa

Several MMS-sensitive mutants of Neurospora crassa were compared with the wild-type strain for their relative sensitivities to UV, X-ray, and histidine. They were also compared for the frequency of spontaneous mutation at the loci which confer resistance to p-fluorophenylalanine. The mutants were also examined for possible defects in meiotic behavior in homozygous crosses and for any change in the inducible DNA salvage pathways (as indicated by their ability to utilize DNA as the sole phosphate source in the growth medium). On the basis of these characterizations, the present MMS-sensitive mutants of Neurospora can be placed into three groups. The first group includes three mutants, mus-(SC3), mus-(SC13), and mus-(SC28). These are slow growers, insensitive to histidine with no apparent meiotic defects and may have reduced frequency of spontaneous mutation. In addition, their mycelial growth is sensitive to MMS but the conidial viability following MMS, UV or X-ray treatment appears normal or only slightly more sensitive than the wild-type. The second group includes only one mutant, mus-(SC15); its mycelial growth is very sensitive to MMS but the conidial survival following treatment with MMS or UV appears normal; however, the conidial survival following exposure to X-ray is significantly reduced. This mutant shows an increase (more than 10-fold) frequency of spontaneous mutation, but behaves normal like the wild-type with respect to fertility, growth rate and insensitivity to histidine. The third group includes mutants mus-(SC10), mus-(SC25), and mus-(SC29). These mutants are very sensitive to UV, X-rays and MMS and to histadine but have normal growth rates on minimal medium. Mutant mus-(SC10), but not mus-(SC25) and mus-(SC29), has an increased (11 ×) frequency of spontaneous mutation. On the basis of data presented, the MMS sensitivity of the first group of mutants cannot be ascertained to arise from a defect in the DNA repair pathways; instead, it may stem from altered cell permeability or other pleotropic effects of the mus mutations. However, it can be suggested that the second and third group of mus mutants may indeed result from a defect in the DNA repair pathways controlled by the mus genes; this conclusion is based on their cross-sensitivity to a number of DNA-damaging agents such as MMS, UV and/or X-ray, high frequencies of spontaneous mutation (mutator effects) and defects in meiotic behavior.     

Induction of telomerase activity by irradiation in human lymphoblasts

Neuhof, D., Ruess, A., Wenz, F. and Weber, K. J. Induction of Telomerase Activity by Irradiation in Human Lymphoblasts. Radiat. Res. 155, 693-697 (2001). Telomerase activity is a radiation-inducible function, which suggests a role of this enzyme in DNA damage processing. Since the tumor suppressor TP53 plays a central role in the regulation of the cellular response to DNA damage, our study explored the ability of ionizing radiation to change telomerase activity and telomere length in two closely related human lymphoblast cell lines with different TP53 status. TK6 cells (wild-type TP53) and WTK1 cells (mutated TP53) were exposed to different doses of X rays, and telomerase activity was measured by PCR ELISA at different times after irradiation. A dose-dependent increase in telomerase activity was observed. One hour after irradiation with 4 Gy, TK6 and WTK1 cells showed an approximately 2.5-fold increase; for lower doses (0.1 to 1 Gy), telomerase induction was seen only in TK6 cells. Telomerase induction was observed by 0.5 h after irradiation, with a further increase up to 24 h. Irradiated TK6 and WTK1 cells had longer telomeres (+1.3 kb) than unirradiated cells 14 days after exposure. Our data demonstrate a dose-dependent induction of telomerase activity and lengthening of telomeres by ionizing radiation in human lymphoblasts. Induction of telomerase activity by radiation does not generally appear to be controlled by the TP53-dependent DNA damage response pathway. However, for low doses, induction of telomerase requires wild-type TP53.     

Phosphorylation of Mcm2 modulates Mcm2-7 activity and affects the cell's response to DNA damage

The S-phase kinase, DDK controls DNA replication through phosphorylation of the replicative helicase, Mcm2-7. We show that phosphorylation of Mcm2 at S164 and S170 is not essential for viability. However, the relevance of Mcm2 phosphorylation is demonstrated by the sensitivity of a strain containing alanine at these positions (mcm2(AA)) to methyl methanesulfonate (MMS) and caffeine. Consistent with a role for Mcm2 phosphorylation in response to DNA damage, the mcm2(AA) strain accumulates more RPA foci than wild type. An allele with the phosphomimetic mutations S164E and S170E (mcm2(EE)) suppresses the MMS and caffeine sensitivity caused by deficiencies in DDK function. In vitro, phosphorylation of Mcm2 or Mcm2(EE) reduces the helicase activity of Mcm2-7 while increasing DNA binding. The reduced helicase activity likely results from the increased DNA binding since relaxing DNA binding with salt restores helicase activity. The finding that the ATP site mutant mcm2(K549R) has higher DNA binding and less ATPase than mcm2(EE), but like mcm2(AA) results in drug sensitivity, supports a model whereby a specific range of Mcm2-7 activity is required in response to MMS and caffeine. We propose that phosphorylation of Mcm2 fine-tunes the activity of Mcm2-7, which in turn modulates DNA replication in response to DNA damage.     

Molecular steps of G‐overhang generation at human telomeres and its function in chromosome end protection

Telomeric G‐overhangs are required for the formation of the protective telomere structure and telomerase action. However, the mechanism controlling G‐overhang generation at human telomeres is poorly understood. Here, we show that G‐overhangs can undergo cell cycle‐regulated changes independent of telomerase activity. G‐overhangs at lagging telomeres are lengthened in S phase and then shortened in late S/G2 because of C‐strand fill‐in, whereas the sizes of G‐overhangs at leading telomeres remain stable throughout S phase and are lengthened in G2/M. The final nucleotides at measurable C‐strands are precisely defined throughout the cell cycle, indicating that C‐strand resection is strictly regulated. We demonstrate that C‐strand fill‐in is mediated by DNA polymerase α (polα) and controlled by cyclin‐dependent kinase 1 (CDK1). Inhibition of CDK1 leads to accumulation of lengthened G‐overhangs and induces telomeric DNA damage response. Furthermore, depletion of hStn1 results in elongation of G‐overhangs and an increase in telomeric DNA damage. Our results suggest that G‐overhang generation at human telomeres is regulated by multiple tightly controlled processes and C‐strand fill‐in is under the control of polα and CDK1.