Tightly bound to DNA proteins: Possible universal substrates for intranuclear processes

Sjakste, Nikolajs; Bielskienė, Kristina; Bagdoniene, L.; Labeikytė, Danutė; Gutcaits, Aleksandrs; Vassetzky, Yegor S.; Sjakste, Tatjana

doi:10.1016/j.gene.2011.09.019

Cited by 7 publications

(6 citation statements)

References 107 publications

(146 reference statements)

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“…These proteins can bind to DNA either through covalent or noncovalent interactions and, even when noncovalently bound, can sometimes withstand high salt and detergent conditions [28]. The wild type HeT-A Gag-FLAG protein when expressed in S2 cells is mostly found in an insoluble nuclear fraction (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…S1), suggesting that the recombinant HeT-A Gag protein was not associated exclusively with either DNA or RNA inside the nucleus. This is not surprising, since a number of other tightly bound proteins, resistant to treatment to nuclease, salts and detergents, are known to be part of the nuclear matrix and are involved in DNA replication, transcription, repair or recombination [28], [29].…”

Section: Resultsmentioning

confidence: 99%

“…Such associations are not common, but a few proteins are known that are tightly bound to the nuclear matrix. These proteins fall under the category of TBPs and can only be isolated with DNase and RNase treatment, followed by extractions with high salt [28]. TBPs include diverse class of proteins such as transcription factors, protein kinases, serpins, and proteins of retrotransposons [31].…”

Section: Resultsmentioning

confidence: 99%

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Phosphorylation at Serines 216 and 221 Is Important for Drosophila HeT-A Gag Protein Stability

et al. 2013

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Telomeres from Drosophila appear to be very different from those of other organisms – in size and the mechanism of their maintenance. In the absence of the enzyme telomerase, Drosophila telomeres are maintained by retrotransposition of three elements, HeT-A, TART, and TAHRE, but details of their transposition mechanisms are not known. Here we characterized some biochemical characteristics of the HeT-A Gag protein encoded by the HeT-A element to understand this mechanism. The HeT-A Gag protein when overexpressed in S2 cells was localized to the nucleus but was resistant to high salt, detergents and nuclease extraction treatments. Analysis of the HeT-A Gag protein by tandem mass spectrophotometry revealed that serines 216 and 221 are phosphorylated. Substituting these serines with alanine or aspartic acid by site-directed mutagenesis did not result in any changes in HeT-A Gag translocation across the nucleus, suggesting that phosphorylation of these sites is not associated with HeT-A Gag translocation, but time course experiments showed that these phosphorylation sites are important for Gag-protein stability.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Phosphorylation at Serines 216 and 221 Is Important for Drosophila HeT-A Gag Protein Stability

et al. 2013

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“…The relationship between TBP-bound DNA and nuclear matrix attachment sites (MARs) has been constantly discussed (Sjakste et al, 2012). To test similarities between our sequences and MARs, we have compared them to sequences deposited in the MAR/SAR database.…”

Section: Similarity To the Mar/sar Sequencesmentioning

confidence: 99%

Tight DNA-protein complexes isolated from barley seedlings are rich in potential guanine quadruplex sequences

Sjakste

Ļeonova

Petrovs

et al. 2020

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Background The concept of chromatin domains attached to the nuclear matrix is being revisited, with nucleus described as a set of topologically associating domains. The significance of the tightly bound to DNA proteins (TBP), a protein group that remains attached to DNA after its deproteinization should be also revisited, as the existence of these interactions is in good agreement with the concept of the topologically associating domain. The work aimed to characterize the DNA component of TBP isolated from barley seedlings. Methods The tight DNA-protein complexes from the first leaves, coleoptiles, and roots of barley seedlings were isolated by purification with chromatography on nitrocellulose or exhaustive digestion of DNA with DNase I. Cloning and transformation were performed using pMOSBBlue Blunt Ended Cloning Kit. Inserts were amplified by PCR, and sequencing was performed on the MegaBace 1000 Sequencing System. The BLAST search was performed using sequence databases at NCBI, CR-EST, and TREP and Ensembl Plants databases. Comparison to MAR/SAR sequences was performed using http://smartdb.bioinf.med.uni-goettingen.de/cgi-bin/SMARtDB/smar.cgi database. The prediction of G quadruplexes (GQ) was performed with the aid of R-studio library pqsfinder. CD spectra were recorded on a Chirascan CS/3D spectrometer. Results Although the barley genome is AT-rich (43% of GC pairs), most DNA fragments associated with TBP were GC-rich (up to 70% in some fractions). Both fractionation procedures yielded a high proportion of CT-motif sequences presented predominantly by the 16-bp CC(TCTCCC)2 TC fragment present in clones derived from the TBP-bound DNA and absent in free DNA. BLAST analysis revealed alignment with different barley repeats. Some clones, however, aligned with both nuclear and chloroplast structural genes. Alignments with MAR/SAR motifs were very few. The analysis produced by the pqsfinder program revealed numerous potential quadruplex-forming sites in the TBP-bound sequences. A set of oligonucleotides containing sites of possible GQs were designed and ordered. Three of them represented the minus strand of the CT-repeat. Two were derived from sequences of two clones of nitrocellulose retained fraction from leaves and contained GC-rich motifs different from the CT motif. Circular dichroism spectroscopy revealed profound changes in spectra when oligonucleotides were incubated with 100 mM KCl. There was either an increase of positive band in the area of 260 nm or the formation of a positive band at 290 nm. In the former case, changes are typical for parallel G-quadruplexes and, in the latter, 3 + 1 structures. Discussion The G-quadruplexes anchor proteins are probably involved in the maintenance of the topologically associated domain structure.

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“…Qualquer um desses fatores pode levar a ativação de um programa genético que culmina na morte celular programada. O gene C1D codifica uma proteína de localização nuclear envolvida em vários processos celulares, tais como transcrição, processamento do RNA, reparo do DNA, morte celular e condensação do cromossomo (SJAKSTE et al, 2012). Apesar de sua presença na célula em níveis fisiológicos ser provavelmente essencial, quando presente em quantidades adicionais leva a morte celular.…”

Section: Apoptoseunclassified

Análise do perfil de expressão gênica de oócitos bovinos advindos de complexos cumulus-oócito com diferentes qualidades morfológicas

Lemes¹

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Tightly bound to DNA proteins: Possible universal substrates for intranuclear processes

Cited by 7 publications

References 107 publications

Phosphorylation at Serines 216 and 221 Is Important for Drosophila HeT-A Gag Protein Stability

Phosphorylation at Serines 216 and 221 Is Important for Drosophila HeT-A Gag Protein Stability

Tight DNA-protein complexes isolated from barley seedlings are rich in potential guanine quadruplex sequences

Análise do perfil de expressão gênica de oócitos bovinos advindos de complexos cumulus-oócito com diferentes qualidades morfológicas

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