Evidence that protein disulfide isomerase (PDI) is involved in DNA–nuclear matrix anchoring

Abstract

DNA-nuclear matrix (NM) anchoring plays a critical role in the organization of DNA within the nucleus and in functional access to DNA for transcription, replication, and DNA repair. The cellular response to oxidative stress involves both gene expression and DNA repair. We, therefore, determined if changes in the oxidative-reductive environment can affect DNA-NM anchoring. The present study used two approaches to study the effect of the reducing agent DTT on DNA-NM anchoring. First, the relative stringency of the DNA-NM attachment was determined by measuring the ability of NM attached DNA loops to undergo supercoiling changes. Second, the effects of DTT on the association of nuclear proteins with DNA were determined by cisplatin crosslinking. When nucleoids (nuclear matrices with attached DNA loops) were prepared from HeLa cells with 1 mM dithiothreitol (DTT), supercoiled DNA loops unwound more efficiently compared with control in the presence of increasing propidium iodide (PI) concentrations. In addition, the rewinding of DNA supercoils in nucleoids treated with DTT was inhibited. Both effects on DNA supercoiling ability were reversed by diamide suggesting that they are dependent on the oxidation state of the protein thiols. When DTT treated nucleoids were isolated from gamma-irradiated cells, the inhibition of DNA supercoil rewinding was equal to the sum of the inhibition due to DTT and gamma-rays alone. Nucleoids isolated from heat-shocked cells with DTT, showed no inhibition of DNA rewinding, except a small inhibition at high PI concentrations. Nuclear DNA in DTT-treated nuclei was digested faster by DNase I than in untreated nuclei. These results suggest that DTT is altering DNA-NM anchoring by affecting the protein component(s) of the anchoring complex. Extracting NM with increasing concentrations of DTT did not solubilize any protein to a significant extent until measurable NM disintegration occurred. Therefore, we determined if 1 mM DTT affected the ability of 1 mM cisplatin to crosslink proteins to DNA. Isolated nuclei were treated with 1 mM DTT for 30 min or left untreated prior to crosslinking with 1 mM cisplatin for 2 h at 4 degrees C. The ability of capsulation to crosslink DNA to proteins per se, did not appear to be affected by 1 mM DTT because relative amounts of at least four proteins, 69, 60, 40, and 35 kDa, were crosslinked to DNA to the same extent in DTT-treated and untreated nuclei. However, protein disulfide isomerase (PDI) crosslinked to DNA in untreated nuclei, but did not crosslink DNA in nuclei that were treated with 1 mM DTT; 1 mM DTT did not affect the intranuclear localization of PDI. Thus, DTT appears to alter the conformation of PDI, as suggested by the DTT-induced change in DNA association, but not its NM association. These results also imply that DNA-NM anchoring involves the redox state of protein sulfhydryl groups.

The protein disulfide isomerases PDIA4 and PDIA6 mediate resistance to cisplatin-induced cell death in lung adenocarcinoma

“…In contrast, PDIA6 can also be detected in the cytoplasm and in the nucleus in some tumor cells in most of the patients. Unusual locations of PDI proteins have been previously proposed to be related either to their redox function or to their ability to bind DNA and nuclear matrix and to activate transcription factors 37,38 or participated to DNA repair. PDI has recently been found in glial cytoplasmic inclusions of patients with multiple system atrophy, 39 Alzheimer's disease 40 and amyotrophic lateral sclerosis.…”

Anaplasma phagocytophilum AnkA binds to granulocyte DNA and nuclear proteins

“…specifically crosslinked to native AnkA by cisdiamminedichloroplatinum (cisplatin or cis -DDP) (Ferraro et al ., 1996;Jenke et al ., 2002;VanderWaal et al ., 2002). When native AnkA was immunoprecipitated from A. phagocytophilum -infected HL-60 cells, DNA was only coimmunoprecipitated from cis-DDP-treated preparations (Fig.…”

“…Although cis-DDP crosslinks proteins that are within 4 Å of DNA at the time of the reaction (VanderWaal et al, 2002), it may not specifically link AnkA to its definitive target DNA binding sequences and instead might allow binding in proximity to the target. Thus, each individual sequence was used as a BLASTN query of both GenBank and the human genome.…”

“…To demonstrate the exclusive presence of AnkA in nuclei and to exclude the possibility of contamination with whole A. phagocytophilum, the protein concentration of the nuclear lysates was measured and identical quantities of the same nuclear prepration were loaded for electrophoresis and immunoblotted for the detection of AnkA and Msp2 protein as above. Immunoblots were also used to demonstrate changes in electrophoretic mobility of AnkA after cis-DDP cross-linking or DNase treatments (Ferraro et al, 1996;Jenke et al, 2002;VanderWaal et al, 2002). For immunoblots, alkaline phosphatase-labelled goat anti-rabbit or mouse IgG (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was used as a secondary antibody.…”

“…To determine if AnkA binds to specific DNA sequences, DNA recovered after AnkA immunoprecipitation from cis-DDP-treated A. phagocytophilum-infected HL-60 cell nuclei was cloned and sequenced (Ferraro et al, 1996;VanderWaal et al, 2002). To do so, DNA fragments of <1500 bp identified by this method were excised and eluted from 0.6% agarose gels (QIAquick Gel Extraction Kit, Qiagen).…”