Improved methods for the generation of human gene knockout and knockin cell lines

Topaloglu, Özlem; Hurley, Paula J.; Yıldırım, Özlem; Civin, Curt I.; Bunz, Fred

doi:10.1093/nar/gni160

Cited by 61 publications

(80 citation statements)

References 18 publications

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“…Endogenous Chk1 alleles were altered in the colorectal cancer cell line DLD-1 by recombinant adeno-associated virus (rAAV)-based gene targeting (30). For the construction of targeting vector homology arms, genomic regions of 1-1.6 kb were amplified from genomic DNA, and sitedirected mutagenesis was used to introduce point mutations within the codons for S317 or S345, thereby converting them into A codons (S317A and S345A, respectively).…”

Section: Methodsmentioning

confidence: 99%

Essential function of Chk1 can be uncoupled from DNA damage checkpoint and replication control

Wilsker

Petermann

Helleday

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

121

131

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Chk1 is widely known as a DNA damage checkpoint signaling protein. Unlike many other checkpoint proteins, Chk1 also plays an essential but poorly defined role in the proliferation of unperturbed cells. Activation of Chk1 after DNA damage is known to require the phosphorylation of several C-terminal residues, including the highly conserved S317 and S345 sites. To evaluate the respective roles of these individual sites and assess their contribution to the functions of Chk1, we used a gene targeting approach to introduce point mutations into the endogenous human CHK1 locus. We report that the essential and nonessential functions of Chk1 are regulated through distinct phosphorylation events and can be genetically uncoupled. The DNA damage response function of Chk1 was nonessential. Targeted mutation of S317 abrogated G 2/M checkpoint activation, prevented subsequent phosphorylation of Chk1, impaired efficient progression of DNA replication forks, and increased fork stalling, but did not impact viability. Thus, the nonessential DNA damage response function of Chk1 could be unambiguously linked to its role in DNA replication control. In contrast, a CHK1 allele with mutated S345 did not support viability, indicating an essential role for this residue during the unperturbed cell cycle. A distinct, physiologic mode of S345 phosphorylation, initiated at the centrosome during unperturbed mitosis was independent of codon 317 status and mechanistically distinct from the ordered and sequential phosphorylation of serine residues on Chk1 induced by DNA damage. Our findings suggest an essential regulatory role for Chk1 phosphorylation during mitotic progression.

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Section: Methodsmentioning

confidence: 99%

Essential function of Chk1 can be uncoupled from DNA damage checkpoint and replication control

Wilsker

Petermann

Helleday

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

121

131

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“…We used recombinant adeno-associated virus (rAAV)-based gene targeting (Topaloglu et al, 2005) to knock-in the point mutation associated with ATR-Seckel syndrome into DLD1 cells. An rAAV delivered a targeting construct carrying the ATR-Seckel mutation A2101G (Figure 1a).…”

Section: Generation Of Atr-seckel Cancer Cells By Homologous Recombinmentioning

confidence: 99%

“…An rAAV was used for gene targeting (Topaloglu et al, 2005). The A2101G mutation found in ATR-Seckel patients was incorporated into a cloned 2.5 kb region of the ATR locus, including exons 9 and 10, by site-directed mutagenesis.…”

Section: Gene Targetingmentioning

confidence: 99%

“…This targeting construct was then subcloned into an rAAV shuttle vector and infectious virus was produced using an AAV helper-free system (Stratagene, La Jolla, CA, USA). Targeted clones were obtained as described previously (Topaloglu et al, 2005).…”

Section: Gene Targetingmentioning

confidence: 99%

“…Using recently developed gene-targeting techniques (Topaloglu et al, 2005), we have introduced a hypomorphic ATR mutation into a human colorectal cancer cell line that harbors mutant p53 alleles and therefore has defective checkpoints. Unexpectedly, we found that in response to IR, checkpoint-deficient cancer cells with hypomorphic ATR alleles failed to progress beyond a critical point in the cell cycle.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Human cancer cells require ATR for cell cycle progression following exposure to ionizing radiation

2006

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The vast majority of cancer cells have defective checkpoints that permit the cell cycle to progress in the presence of double-strand DNA breaks (DSBs) caused by ionizing radiation (IR) and radiomimetic drugs. ATR (ataxia telangiectasia-mutated and Rad3-related) has recently been shown to be activated by DSBs, although the consequences of this activity are largely unknown. In this report, we use advanced gene targeting methods to generate biallelic hypomorphic ATR mutations in human colorectal cancer cells and demonstrate that progression of the cancer cell cycle after IR treatment requires ATR. Cells with mutant ATR accumulated at a defined point at the beginning of the S phase after IR treatment and were unable to progress beyond that point, whereas cells at later stages of the S phase during the time of irradiation progressed and completed DNA replication. The prolonged arrest of ATR mutant cancer cells did not involve the ataxia telangiectasia mutated-dependent S-phase checkpoint, but rather closely resembled a previously characterized form of cell cycle arrest termed S-phase stasis. As ATR strongly contributed to clonogenic survival after IR treatment, these data suggest that blocking ATR activity might be a useful strategy for inducing S-phase stasis and promoting the radiosensitization of checkpointdeficient cancer cells.

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Tumor‐associated APE1 variant exhibits reduced complementation efficiency but does not promote cancer cell phenotypes

Illuzzi

McNeill

Bastian

et al. 2017

Environ and Mol Mutagen

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Base excision repair (BER) is the major pathway for coping with most forms of endogenous DNA damage, and defects in the process have been associated with carcinogenesis. Apurinic/apyrimidinic endonuclease 1 (APE1) is a central participant in BER, functioning as a critical endonuclease in the processing of non-coding abasic sites in DNA. Evidence has suggested that APE1 missense mutants, as well as altered expression or localization of the protein, can contribute to disease manifestation. We report herein that the tumor-associated APE1 variant, R237C, shows reduced complementation efficiency of the methyl methanesulfonate hypersensitivity and impaired cell growth exhibited by APE1-deficient mouse embryonic fibroblasts. Overexpression of wild-type APE1 or the R237C variant in the non-transformed C127I mouse cell line had no effect on proliferation, cell cycle status, steady-state DNA damage levels, mitochondrial function or cellular transformation. A human cell line heterozygous for an APE1 knockout allele had lower levels of endogenous APE1, increased cellular sensitivity to DNA-damaging agents, impaired proliferation with time, and a distinct global gene expression pattern consistent with a stress phenotype. Our results indicate that: (i) the tumor-associated R237C variant is a possible susceptibility factor, but not likely a driver of cancer cell phenotypes, (ii) overexpression of APE1 does not readily promote cellular transformation, and (iii) haploinsufficiency at the APE1 locus can have profound cellular consequences, consistent with BER playing a critical role in proliferating cells.

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Improved methods for the generation of human gene knockout and knockin cell lines

Cited by 61 publications

References 18 publications

Essential function of Chk1 can be uncoupled from DNA damage checkpoint and replication control

Essential function of Chk1 can be uncoupled from DNA damage checkpoint and replication control

Human cancer cells require ATR for cell cycle progression following exposure to ionizing radiation

Tumor‐associated APE1 variant exhibits reduced complementation efficiency but does not promote cancer cell phenotypes

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