The impact of a His-tag on DNA binding by RNA polymerase alpha-C-terminal domain from Helicobacter pylori

Paul, Navjit K.; Baksh, Karina A.; Arias, Joaquin F.; Zamble, Deborah B.

doi:10.1016/j.pep.2019.105541

Cited by 13 publications

(9 citation statements)

References 48 publications

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“…Importantly, upon removal of the His-tag used for purification purposes, the binding of RAP1 to free and nucleosome-bound DNA was abrogated (Figure 2G; Figure S2G-H). The confounding impact of histidine tags on DNAbinding activity of purified proteins has been reported in the literature (Paul et al 2020) and could well explain earlier studies that showed nonspecific binding of human RAP1 to DNA in vitro (Arat and Griffith 2012). Taken together, our data confirm that unlike budding yeast Rap1, the mammalian counterpart is incapable of binding DNA directly, and therefore, the mechanism by which RAP1 regulates gene expression in higher eukaryotes must have diverged from budding yeast.…”

Section: Rap1 I312r Does Not Directly Associate With Chromatinsupporting

confidence: 88%

RAP1 regulates TIP60 function during fate transition between 2 cell-like and pluripotent states

Barry

Sacco

Mameri

et al. 2021

Preprint

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SummaryIn mammals, the conserved telomere binding protein RAP1 serves a diverse set of non- telomeric functions including activation of the NF-kB signaling pathway, maintenance of metabolic function in vivo, and transcriptional regulation. Here, we uncover the mechanism by which RAP1 modulates gene expression. Using a separation-of-function allele, we show that RAP1 transcriptional regulation is independent of TRF2-mediated binding to telomeres and does not involve direct binding to genomic loci. Instead, RAP1 interacts with the TIP60/p400 complex and modulates its histone acetyltransferase activity. Notably, we show that deletion of RAP1 in mouse embryonic stem cells increases the fraction of 2-cell-like cells. Specifically, RAP1 enhances the repressive activity of Tip60/p400 across a subset of 2-cell-stage genes, including Zscan4 and the endogenous retrovirus MERVL. Preferential upregulation of genes proximal to MERVL elements in Rap1 deficient settings implicate these endogenous retroviral elements in the de- repression of proximal genes. Altogether, our study reveals an unprecedented link between RAP1 and TIP60/p400 complex in the regulation of totipotency.

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Section: Rap1 I312r Does Not Directly Associate With Chromatinsupporting

confidence: 88%

RAP1 regulates TIP60 function during fate transition between 2 cell-like and pluripotent states

Barry

Sacco

Mameri

et al. 2021

Preprint

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show abstract

“…S3G,H ). The confounding impact of histidine tags on DNA-binding activity of purified proteins has been previously reported ( Paul et al 2020 ) and could well explain earlier studies that showed nonspecific binding of human RAP1 to DNA in vitro ( Arat and Griffith 2012 ). Taken together, our data confirm that, unlike budding yeast Rap1, the mammalian counterpart is incapable of binding DNA directly, and therefore the mechanisms by which RAP1 regulates gene expression in higher eukaryotes diverged.…”

Section: Resultssupporting

confidence: 61%

Rap1 regulates TIP60 function during fate transition between two-cell-like and pluripotent states

et al. 2022

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In mammals, the conserved telomere binding protein Rap1 serves a diverse set of nontelomeric functions, including activation of the NF-kB signaling pathway, maintenance of metabolic function in vivo, and transcriptional regulation. Here, we uncover the mechanism by which Rap1 modulates gene expression. Using a separation-of-function allele, we show that Rap1 transcriptional regulation is largely independent of TRF2-mediated binding to telomeres and does not involve direct binding to genomic loci. Instead, Rap1 interacts with the TIP60/p400 complex and modulates its histone acetyltransferase activity. Notably, we show that deletion of Rap1 in mouse embryonic stem cells increases the fraction of two-cell-like cells. Specifically, Rap1 enhances the repressive activity of Tip60/p400 across a subset of two-cell-stage genes, including Zscan4 and the endogenous retrovirus MERVL. Preferential up-regulation of genes proximal to MERVL elements in Rap1-deficient settings implicates these endogenous retroviral elements in the derepression of proximal genes. Altogether, our study reveals an unprecedented link between Rap1 and the TIP60/p400 complex in the regulation of pluripotency.

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“…The pET24bhpnikRG27 vector was generated as previously described ( 95 ) and used as the template to prepare the HpNikR mutants. Site-directed mutagenesis was performed according to the QuikChange site-directed mutagenesis protocol (Stratagene) by using primers listed in Table 3 , to generate the HpNikR mutants, except K64A/K140A.…”

Section: Methodsmentioning

confidence: 99%

Mechanistic insights into the nickel-dependent allosteric response of the Helicobacter pylori NikR transcription factor

Baksh

Augustine

Sljoka

et al. 2023

Journal of Biological Chemistry

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The impact of a His-tag on DNA binding by RNA polymerase alpha-C-terminal domain from Helicobacter pylori

Cited by 13 publications

References 48 publications

RAP1 regulates TIP60 function during fate transition between 2 cell-like and pluripotent states

RAP1 regulates TIP60 function during fate transition between 2 cell-like and pluripotent states

Rap1 regulates TIP60 function during fate transition between two-cell-like and pluripotent states

Mechanistic insights into the nickel-dependent allosteric response of the Helicobacter pylori NikR transcription factor

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