Distinct patterns of MCM protein binding in nuclei of S phase and rereplicating SV40‐infected monkey kidney cells

Friedrich, Thomas; Bedner, Elżbieta; Darzynkiewicz, Zbigniew; Lehman, John M.

doi:10.1002/cyto.a.20185

Cited by 13 publications

(17 citation statements)

References 55 publications

(54 reference statements)

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“…We used a flow cytometric approach providing a direct and quantitative measurement of global chromatin-bound levels of MCMs throughout cell cycle progression without cell synchronization. 26 A representative contour plot of chromatin-bound MCM3 versus DNA content (4Ј,6-diamidino-2-phenylindole) is shown in Figure 2A. Labeling of chromatin-bound MCM2 yielded similar patterns (data not shown).…”

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confidence: 55%

Interaction of HTLV-1 Tax with minichromosome maintenance proteins accelerates the replication timing program

Boxus

Twizere

Legros

et al. 2012

Blood

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The Tax oncoprotein encoded by the human T-cell leukemia virus type 1 plays a pivotal role in viral persistence and pathogenesis. Human T-cell leukemia virus type 1-infected cells proliferate faster than normal lymphocytes, expand through mitotic division, and accumulate genomic lesions. Here, we show that Tax associates with the minichromosome maintenance MCM2-7 helicase complex and localizes to origins of replication. Tax IntroductionHuman T-cell leukemia virus-1 (HTLV-1) is a retrovirus that infects approximately 20 million people worldwide. 1 Although the majority of infected people remain lifelong asymptomatic carriers, 2% to 5% develop either adult T-cell leukemia (ATL) or chronic inflammatory diseases, such as HTLV-1-associated myelopathy/tropical spastic paraparesis. 1 Two major hallmarks of HTLV-1-infected cells are: preferential proliferation compared with normal lymphocytes 2 and tendency to accumulate genomic lesions, a prerequisite for oncogenesis. 3,4 Although the molecular mechanisms underlying T-cell transformation remain to be elucidated, it is generally accepted that the virally encoded Tax protein plays a central role. Tax indeed displays pleiotropic activities that cooperate to drive cell cycle progression and perturb genome stability. 4,5 For example, Tax shortens G 1 phase and accelerates transition into S phase by manipulating positive and negative G 1 phase regulators (ie, cyclin D/CDK complex, Rb and CDK inhibitors). [6][7][8] In addition, Tax is thought to indirectly generate DNA damage by suppressing DNA repair processes and competing with cellular DNA damage response (DDR). 4,5 Recent data also support a direct role for Tax-induced reactive oxygen species in the occurrence of DNA lesions. 9 Conceptually, Tax activities are achieved through proteinprotein interactions in both nuclear and cytoplasmic cellular compartments. 10 In the nucleus, Tax accumulates within foci called Tax speckled structures (TSS), 11 which overlap with the cellular machinery required for transcription, splicing, post-transcriptional modifications of proteins, DNA repair, and checkpoint control. 12 In this report, we further demonstrate that Tax recruits the MCM2-7 replicative helicase within the TSS and modulates the program of replication origin firing. Notably, this mechanism favors cell cycle progression and directly compromises genome stability. Methods Plasmids, antibodies, yeast 2-hybrid, and GST pulldownSee supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Cell cultureHeLa, Rat-1, and 293GP2 cells were cultured in Dulbecco modified Eagle medium supplemented with 10% FBS and 2mM l-glutamine and penicillin/ streptomycin. Jurkat, HUT78 and JPX9 (Jurkat cell lines stably transfected either with tax-1 gene driven by a metallothionein-inducible promoter) were kept in RPMI 1640 supplemented with 10% FBS, 2mM l-glutamine, and penicillin/streptomycin. Expression of the viral protein was induced in JPX9 cells by adding 120 M zinc chloride...

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confidence: 55%

Interaction of HTLV-1 Tax with minichromosome maintenance proteins accelerates the replication timing program

Boxus

Twizere

Legros

et al. 2012

Blood

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“…This protein is virally encoded and is the only factor required for replication (63). It functions as a replicative helicase allowing replication of SV40 independently from genomic replication and is uncoupled from the ''once per cell cycle'' licensing (64,65). Hence, SV40 replicon-based vectors exhibit high copy numbers (several thousands) per transfected cell (66).…”

Section: Epigenetic Regulation In Episomally Replicating Vectors Viramentioning

confidence: 98%

The epigenetic regulation of autonomous replicons

Hagedorn¹,

Lipps²,

Rupprecht³

2010

BioMolecular Concepts

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The discovery of autonomous replicating sequences (ARSs) in Saccharomyces cerevisiae in 1979 was considered a milestone in unraveling the regulation of replication in eukaryotic cells. However, shortly afterwards it became obvious that in Saccharomyces pombe and all other higher organisms ARSs were not sufficient to initiate independent replication. Understanding the mechanisms of replication is a major challenge in modern cell biology and is also a prerequisite to developing application-oriented autonomous replicons for gene therapeutic treatments. This review will focus on the development of non-viral episomal vectors, their use in gene therapeutic applications and our current knowledge about their epigenetic regulation.

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“…These patterns were first shown by Friedrich and co-workers [17] and are based on measuring the residue of Mcm proteins that are left behind after detergent extraction. In support of this idea, Mukherjee at al.…”

Section: Introductionmentioning

confidence: 94%

“…The reported cytometric pattern of Mcm2 and DNA content for CV-1 cells provides a visualization of Mcm binding in G1 and Mcm removal from chromatin during S phase [17]. The expression of detergent resistant Mcm2 was bimodal in G1 with low and high expressing cell clusters.…”

Section: Introductionmentioning

confidence: 99%

Cytometry of chromatin bound Mcm6 and PCNA identifies two states in G1 that are separated functionally by the G1 restriction point1

Frisa

Jacobberger

2010

BMC Cell Biol

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BackgroundCytometric measurements of DNA content and chromatin-bound Mcm2 have demonstrated bimodal patterns of expression in G1. These patterns, the replication licensing function of Mcm proteins, and a correlation between Mcm loading and cell cycle commitment for cells re-entering the cell cycle, led us to test the idea that cells expressing a defined high level of chromatin-bound Mcm6 in G1 are committed - i.e., past the G1 restriction point. We developed a cell-based assay for tightly-bound PCNA (PCNA*) and Mcm6 (Mcm6*), DNA content, and a mitotic marker to clearly define G1, S, G2, and M phases of the cell cycle. hTERT-BJ1, hTERT-RPE-1, and Molt4 cells were extracted with Triton X-100 followed by methanol fixation, stained with antibodies and DAPI, then measured by cytometry.ResultsBivariate analysis of cytometric data demonstrated complex patterns with distinct clustering for all combinations of the 4 variables. In G1, cells clustered in two groups characterized by low and high Mcm6* expression. Serum starvation and release experiments showed that residence in the high group was in late G1, just prior to S phase. Kinetic experiments, employing serum withdrawal, and stathmokinetic analysis with aphidicolin, mimosine or nocodazole demonstrated that cells with high levels of Mcm6* cycled with the committed phases of the cell cycle (S, G2, and M).ConclusionsA multivariate assay for Mcm6*, PCNA*, DNA content, and a mitotic marker provides analysis capable of estimating the fraction of pre and post-restriction point G1 cells and supports the idea that there are at least two states in G1 defined by levels of chromatin bound Mcm proteins.

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Distinct patterns of MCM protein binding in nuclei of S phase and rereplicating SV40‐infected monkey kidney cells

Cited by 13 publications

References 55 publications

Interaction of HTLV-1 Tax with minichromosome maintenance proteins accelerates the replication timing program

Interaction of HTLV-1 Tax with minichromosome maintenance proteins accelerates the replication timing program

The epigenetic regulation of autonomous replicons

Cytometry of chromatin bound Mcm6 and PCNA identifies two states in G1 that are separated functionally by the G1 restriction point1

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