Cytogenetic analysis of spontaneous and 2‐cyanoethylene oxide‐induced <i>tk</i> —/— mutants in TK6 human lymphoblastoid cultures

Kodama, Y.; Boreiko, Craig J.; Skopek, Thomas R.; Recio, Leslie

doi:10.1002/em.2850140304

Cited by 14 publications

(5 citation statements)

References 23 publications

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“…), t(21;?). These karyotypic features are essentially identical with those found in previous analyses (15,21). On the other hand, WTK-1 commonly showed 47, XY, 13ϩ, t(21;?…”

Section: Resultssupporting

confidence: 87%

Illegitimate Recombination Leading to Allelic Loss and Unbalanced Translocation in p53-Mutated Human Lymphoblastoid Cells

Honma¹,

Zhang²,

Hayashi³

et al. 1997

Molecular and Cellular Biology

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Allelic loss and translocation are critical mutational events in human tumorigenesis. Allelic loss, which is usually identified as loss of heterozygosity (LOH), is frequently observed at tumor suppressor loci in various kinds of human tumors. It is generally thought to result from deletion or mitotic recombination between homologous chromosomes. In this report, we demonstrate that illegitimate (nonhomologous) recombination strongly contributes to the generation of allelic loss in p53-mutated cells. Spontaneous and X-ray-induced LOH mutations at the heterozygous thymidine kinase (tk) gene, which is located on the long arm of chromosome 17, from normal (TK6) and p53-mutated (WTK-1) human lymphoblastoid cells were cytogenetically analyzed by chromosome 17 painting. We observed unbalanced translocations in 53% of LOH mutants spontaneously arising from WTK-1 cells but none spontaneously arising from TK6 cells. We postulate that illegitimate recombination was occurring between nonhomologous chromosomes after DNA replication, leading to allelic loss and unbalanced translocations in p53-mutated WTK-1 cells. X-ray irradiation, which induces DNA double-strand breaks (DSBs), enhanced the generation of unbalanced translocation more efficiently in WTK-1 than in TK6 cells. This observation implicates the wild-type p53 protein in the regulation of homologous recombination and recombinational DNA repair of DSBs and suggests a possible mechanism by which loss of p53 function may cause genomic instability.

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“…), t(21;?). These karyotypic features are essentially identical with those found in previous analyses (15,21). On the other hand, WTK-1 commonly showed 47, XY, 13ϩ, t(21;?…”

Section: Resultssupporting

confidence: 87%

Illegitimate Recombination Leading to Allelic Loss and Unbalanced Translocation in p53-Mutated Human Lymphoblastoid Cells

Honma¹,

Zhang²,

Hayashi³

et al. 1997

Molecular and Cellular Biology

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“…A and B). This result conforms to a previous reported karyotype for TK6 [Kodama et al, ]. FISH was performed using a probe for the Smith–Magenis syndrome deletion region (17p11) which is approximately 2 × 10 6 basepairs 3′ (centromeric) to the PIGL gene.…”

Section: Resultssupporting

confidence: 89%

Both PIGA and PIGL mutations cause GPI‐a deficient isolates in the Tk6 cell line

Nicklas

Carter

Albertini

2015

Environ and Mol Mutagen

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Molecular analysis of proaerolysin selected glycosylphosphatidylinositol anchor (GPI-a) deficient isolates in the TK6 cell line was performed. Initial studies found that the expected X-linked PIGA mutations were rare among the spontaneous isolates but did increase modestly after ethyl methane sulfate (EMS) treatment (but to only 50% of isolates). To determine the molecular bases of the remaining GPI-a deficient isolates, real-time analysis for all the 25 autosomal GPI-a pathway genes was performed on the isolates without PIGA mutations, determining that PIGL mRNA was absent for many. Further analysis determined these isolates had several different homozygous deletions of the 5’ region of PIGL (17p12-p22) extending 5’ (telomeric) through NCOR1 and some into the TTC19 gene (total deletion >250,000bp). It was determined that the TK6 parent had a hemizygous deletion in 17p12-p22 (275,712bp) extending from PIGL intron 2 into TTC19 intron 7. Second hit deletions in the other allele in the GPI-a deficient isolates led to the detected homozygous deletions. Several of the deletion breakpoints including the original first hit deletion were sequenced. As strong support for TK6 having a deletion, a number of the isolates without PIGA mutations nor homozygous PIGL deletions had point mutations in the PIGL gene. These studies show that the GPI-a mutation studies using TK6 cell line could be a valuable assay detecting point and deletion mutations in two genes simultaneously.

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“…The selected clone did not express PRIMPOL protein ( Supplementary Figure S1B ). TK6 cells are frequently used to study DNA replication and the response to DNA damage in human due to their karyotypic stability ( 58 , 59 ) and their expression of functional p53 ( 60 ). To facilitate CRISPR/Cas9 screens, expression cassettes allowing stable expression of Cas9, YFP and blasticidin S deaminase (BSD) were inserted into the AAVS1 safe harbour locus ( 61 ) of both TK6 WT and primpol cells ( Supplementary Figure S1C and D ).…”

Section: Resultsmentioning

confidence: 99%

PRIMPOL ensures robust handoff between on-the-fly and post-replicative DNA lesion bypass

Mellor,

Nassar,

Šviković

et al. 2023

Nucleic Acids Research

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The primase/polymerase PRIMPOL restarts DNA synthesis when replication is arrested by template impediments. However, we do not have a comprehensive view of how PRIMPOL-dependent repriming integrates with the main pathways of damage tolerance, REV1-dependent ‘on-the-fly’ lesion bypass at the fork and PCNA ubiquitination-dependent post-replicative gap filling. Guided by genome-wide CRISPR/Cas9 screens to survey the genetic interactions of PRIMPOL in a non-transformed and p53-proficient human cell line, we find that PRIMPOL is needed for cell survival following loss of the Y-family polymerases REV1 and POLη in a lesion-dependent manner, while it plays a broader role in promoting survival of cells lacking PCNA K164-dependent post-replicative gap filling. Thus, while REV1- and PCNA K164R-bypass provide two layers of protection to ensure effective damage tolerance, PRIMPOL is required to maximise the effectiveness of the interaction between them. We propose this is through the restriction of post-replicative gap length provided by PRIMPOL-dependent repriming.

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Cytogenetic analysis of spontaneous and 2‐cyanoethylene oxide‐induced tk —/— mutants in TK6 human lymphoblastoid cultures

Cited by 14 publications

References 23 publications

Illegitimate Recombination Leading to Allelic Loss and Unbalanced Translocation in p53-Mutated Human Lymphoblastoid Cells

Illegitimate Recombination Leading to Allelic Loss and Unbalanced Translocation in p53-Mutated Human Lymphoblastoid Cells

Both PIGA and PIGL mutations cause GPI‐a deficient isolates in the Tk6 cell line

PRIMPOL ensures robust handoff between on-the-fly and post-replicative DNA lesion bypass

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