Formaldehyde crosslinking protein dna relationship

formaldehyde crosslinking protein dna relationship

capturing site-specific protein–DNA interactions. Extending such these relationships. of formaldehyde crosslinking to model chromatin-. Glycine is commonly used to quench formaldehyde cross-linking reactions .. protein and DNA can preserve the specific binding relationship. Background: Formaldehyde (FA) is classified as a probable human carcinogen. Aims: To examine DNA protein crosslinks (DPC) and p53, which are generally.

Formaldehyde crosslinks DNA-binding proteins such as histones and transcription factors to DNA in vivo and in vitro, whereas free protein is not crosslinked to DNA in vitro, even when present at high concentrations 11 Although in vivo formaldehyde crosslinking is routinely used in conjunction with immunoprecipitation techniques to identify DNA sequences bound by specific proteins 9it also has been used to recover proteins associated with kinetoplast DNA of trypanosomes We describe here the identification of 11 proteins 10 by mass spectrometry that are formaldehyde crosslinked to mtDNA in organello.

Some of these proteins have been shown previously to interact with mtDNA or to function in mtDNA maintenance, but others, such as the mitochondrial chaperone Hsp60p and the tricarboxylic acid TCA cycle protein Kgd2p, were not previously known to interact with mtDNA. Biochemical experiments show, surprisingly, that Hsp60p is a single-stranded ss DNA binding protein that binds specifically and with strand specificity to a putative origin of mtDNA replication.

Materials and Methods Yeast Strains and Media. Purification of Mitochondria and Formaldehyde Crosslinked Nucleoids.

An Improved Method for Measuring Chromatin-binding Dynamics

Cells were washed with SCE, pH 7. The reaction was quenched with mM glycine, pH 7. Some experiments included a second CsCl gradient. Cytotoxicity of FA treatments was determined by colony formation assay. Approximately 2 weeks later colonies were fixed with methanol and stained with Giemsa solution for counting.

DNA—protein crosslinking assay The procedure for measurement of DPC was based on the earlier published protocol 24 with some modifications. DNA was measured in the presence of 0. Fluorescent measurements were recorded 15 min after addition of the dye emission nm, excitation nm. All manipulations with PicoGreen-containing samples were done under reduced illumination. In cultured cells DPC were determined by measuring [3H]thymidine radioactivity of the pellet and supernatant fractions.

The number of crosslinks per bp was calculated from the fraction of DNA fragments containing crosslinks and from the size of DNA fragments. It was important to determine the weighted average length of DNA fragments, which was calculated from the computer scanned images of agarose gels. L is measured in DNA base pairs. Digitized profiles of ethidium bromide stained gels acquired on a GelDoc photodocumentation system Bio-Rad were transferred to Microsoft Excel and the weighted average length was calculated.

From DNA to protein - 3D

A typical DNA fragment size for monolayer cell lines was in the range 20—23 kb. The following equation was used: The R2 values were never less than 0.

formaldehyde crosslinking protein dna relationship

Details of determination of the rate of spontaneous loss of DPC kpassive are given in Results. Due to high cytotoxicity at later time points, a single point was used to calculate the half-life in XP-A fibroblasts treated with lactacystin. The following formula was used: The procedure was adopted from Newton and Fahey GSH-containing supernatants were obtained after centrifugation at 12 g for 10 min at room temperature.

The reaction was allowed to proceed for 10 min in the dark at room temperature and was then terminated by addition of 25 mM methanesulfonic acid.

The specificity of protein–DNA crosslinking by formaldehyde: in vitro and in Drosophila embryos

The aqueous solvent was 0. The GSH peak was eluted at Depletion of GSH in A cells was achieved by adding 0.

This treatment with BSO has been previously shown to be non-toxic to cells Results Rate of spontaneous loss of DNA—histone crosslinks Previous studies have shown that aldehyde-induced DPC were hydrolytically labile and could be completely reversed by incubation at elevated temperatures 22 Our first experiments were performed to determine the rate of spontaneous hydrolysis of FA-induced DPC at physiological temperature and pH.

The intrinsic instability of DPC was examined in crosslinks produced in cells and with in vitro formed DNA—histone crosslinks. Analysis of DPC stability in cell samples could be hampered by the presence of nucleases and proteases, activation of which could lead to an apparent loss of DPC. DNA—histone H1 crosslinks produced in vitro are free of hydrolytic enzymes, but they need to resemble those formed in cells. Figure 1 shows that the time courses of DPC formation by FA in vitro with histone H1 and in cultured A cells were very close, suggesting involvement of similar chemical groups in crosslink formation.

It has been previously shown that 0. The half-life was The primary reason for the addition of SDS to the crosslink samples was to block nuclease activity in cell lysates.

The presence of SDS also prevents reformation of broken crosslinks, since histones will immediately dissociate from DNA after crosslink rupture. This suggests that under physiological conditions a fraction of previously broken DPCs between histones and DNA reform. DPC removal from control and GSH-depleted cells The rate of DPC loss was determined by measuring the number of remaining crosslinks at different time intervals following removal of FA-containing medium.

Two observations provided a basis for these experiments. It has also been reported that human lymphocytes contain lower amounts of GSH than other cells 29 Based on the reported volume of A cells 3. Long-term survival of GSH-depleted and control cells exposed to different concentrations of FA was not statistically different Figure 6B.

Figure 7A shows that the kinetics of DPC removal were very similar among all three cell lines. These results indicate that the nucleotide excision pathway is unlikely to be involved in repair of DNA—histone crosslinks.

formaldehyde crosslinking protein dna relationship

Effect of a proteosome inhibitor lactacystin on repair of DPC Cells eliminate damaged or unnecessary proteins through a specialized proteolytic process Protein targets are first tagged by ubiquitination and then cleaved by a high molecular weight protease complex proteosomes.

We examined the possibility that this normal process of protein degradation is involved in elimination of DPC. Lactacystin is a microbial product that has been shown to be a very potent and specific inhibitor of the proteosome This compound inhibits all three major peptidase activities of the proteosome: Addition of micromolar concentrations of lactacystin resulted in a dose-dependent inhibition of DPC repair in both normal and XP-A fibroblasts Figure 8.

This concentration of lactacystin has been shown to block the proteolytic function of nuclear proteosomes Prolonged exposure to lactacystin resulted in cytotoxicity, evident as accelerated detachment of cells from the dishes. Discussion The main goal of this investigation was to identify major factors that control removal of DPC formed by FA. We initially set out to determine whether spontaneous loss of DPC could represent one of the important contributors to the overall rate of DPC repair in cells.

DNA—histone H1 crosslinks were used in all in vitro experiments, largely due to concerns about the presence of hydrolytic enzymes in DPC preparations from exposed cells. We believe that crosslinks with histone H1 represent a typical DPC from cells because: The rate of DPC disappearance from cells was almost twice as fast as the estimated hydrolytic instability. This suggests that cells also possess an active repair process for DPC removal.

Half-life values were close among three different cell types fibroblasts and kidney and lung cells. This translates into three times less efficient removal of DPC by the active repair component Lymphocytes are also known to have inefficient nucleotide excision repair Lymphocytes are terminally differentiated cells and, because there is no danger of converting DNA lesions into mutations, the presence of active DNA repair in these cells may not be biologically important.

Damaged lymphocytes can also be easily replaced through production of new cells.