DNA fragmentation

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DNA fragmentation is the separation or breaking of DNA strands into pieces. It can be done intentionally by laboratory personnel or by cells, or can occur spontaneously. Spontaneous or accidental DNA fragmentation is fragmentation that gradually accumulates in a cell. It can be measured by e.g. the Comet assay or by the TUNEL assay.

Contents

Its main units of measurement is the DNA Fragmentation Index (DFI). [1] A DFI of 20% or more significantly reduces the success rates after ICSI. [1]

DNA fragmentation was first documented by Williamson in 1970 when he observed discrete oligomeric fragments occurring during cell death in primary neonatal liver cultures. He described the cytoplasmic DNA isolated from mouse liver cells after culture as characterized by DNA fragments with a molecular weight consisting of multiples of 135 kDa. This finding was consistent with the hypothesis that these DNA fragments were a specific degradation product of nuclear DNA. [2]

Intentional

DNA fragmentation is often necessary prior to library construction or subcloning for DNA sequences. A variety of methods involving the mechanical breakage of DNA have been employed where DNA is fragmented by laboratory personnel. Such methods include sonication, needle shear, nebulisation, point-sink shearing and passage through a pressure cell. [3]

Spontaneous

Apoptotic DNA fragmentation is a natural fragmentation that cells perform in apoptosis (programmed cell death). DNA fragmentation is a biochemical hallmark of apoptosis. In dying cells, DNA is cleaved by an endonuclease that fragments the chromatin into nucleosomal units, which are multiples of about 180-bp oligomers and appear as a DNA ladder when run on an agarose gel. [8] The enzyme responsible for apoptotic DNA fragmentation is the Caspase-activated DNase. CAD is normally inhibited by another protein, the Inhibitor of Caspase Activated DNase (ICAD). During apoptosis, the apoptotic effector caspase, caspase 3, cleaves ICAD and thus causes CAD to become activated. [9]

A nucleosome, consisting of DNA (grey) wrapped around a histone tetramer (coloured). In apoptotic DNA fragmentation, the DNA is cleaved in the internucleosomal linker region, which is the part of the DNA not wrapped around the histones. Complete Histone with DNA.png
A nucleosome, consisting of DNA (grey) wrapped around a histone tetramer (coloured). In apoptotic DNA fragmentation, the DNA is cleaved in the internucleosomal linker region, which is the part of the DNA not wrapped around the histones.

CAD cleaves the DNA at the internucleosomal linker sites between the nucleosomes, protein-containing structures that occur in chromatin at ~180-bp intervals. This is because the DNA is normally tightly wrapped around histones, the core proteins of the nucleosomes. The linker sites are the only parts of the DNA strand that are exposed and thus accessible to CAD.

Men with sperm motility defects often have high levels of sperm DNA fragmentation. [10] The degree of DNA fragmentation in sperm cells can predict outcomes for in vitro fertilization [11] (IVF) and its expansion intracytoplasmic sperm injection [1] (ICSI). The sperm chromatin dispersion test (SCD) and TUNEL assay are both effective in detecting sperm DNA damage. [12] [13] Using bright-field microscopy, the SCD test appears to be more sensitive than the TUNEL assay. [13]

Uses

DNA Fragmentation plays an important part in forensics, especially that of DNA profiling.

Related Research Articles

<span class="mw-page-title-main">Apoptosis</span> Programmed cell death in multicellular organisms

Apoptosis is a form of programmed cell death that occurs in multicellular organisms and in some eukaryotic, single-celled microorganisms such as yeast. Biochemical events lead to characteristic cell changes (morphology) and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, DNA fragmentation, and mRNA decay. The average adult human loses between 50 and 70 billion cells each day due to apoptosis. For an average human child between eight and fourteen years old, each day the approximate lost is 20 to 30 billion cells.

Deoxyribonuclease refers to a group of glycoprotein endonucleases which are enzymes that catalyze the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. The role of the DNase enzyme in cells includes breaking down extracellular DNA (ecDNA) excreted by apoptosis, necrosis, and neutrophil extracellular traps (NET) of cells to help reduce inflammatory responses that otherwise are elicited. A wide variety of deoxyribonucleases are known and fall into one of two families, which differ in their substrate specificities, chemical mechanisms, and biological functions. Laboratory applications of DNase include purifying proteins when extracted from prokaryotic organisms. Additionally, DNase has been applied as a treatment for diseases that are caused by ecDNA in the blood plasma. Assays of DNase are emerging in the research field as well.

<span class="mw-page-title-main">Karyorrhexis</span> Destructive fragmentation of the nucleus of a dying cell

Karyorrhexis is the destructive fragmentation of the nucleus of a dying cell whereby its chromatin is distributed irregularly throughout the cytoplasm. It is usually preceded by pyknosis and can occur as a result of either programmed cell death (apoptosis), cellular senescence, or necrosis.

<span class="mw-page-title-main">TUNEL assay</span>

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) is a method for detecting DNA fragmentation by labeling the 3′- hydroxyl termini in the double-strand DNA breaks generated during apoptosis.

<span class="mw-page-title-main">DNA laddering</span>

DNA laddering is a feature that can be observed when DNA fragments, resulting from Apoptosis DNA fragmentation are visualized after separation by gel electrophoresis the first described in 1980 by Andrew Wyllie at the University Edinburgh medical school DNA fragments can also be delected in cells that underwent necrosis, when theses DNA fragments after separation are subjected to gel electrophoresis which in the results in a characteristic ladder pattern,

<span class="mw-page-title-main">Deoxyribonuclease I</span> Protein-coding gene in the species Homo sapiens

Deoxyribonuclease I, is an endonuclease of the DNase family coded by the human gene DNASE1. DNase I is a nuclease that cleaves DNA preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide, yielding 5'-phosphate-terminated polynucleotides with a free hydroxyl group on position 3', on average producing tetranucleotides. It acts on single-stranded DNA, double-stranded DNA, and chromatin. In addition to its role as a waste-management endonuclease, it has been suggested to be one of the deoxyribonucleases responsible for DNA fragmentation during apoptosis.

<span class="mw-page-title-main">Apoptosis-inducing factor</span> Protein family

Apoptosis inducing factor is involved in initiating a caspase-independent pathway of apoptosis by causing DNA fragmentation and chromatin condensation. Apoptosis inducing factor is a flavoprotein. It also acts as an NADH oxidase. Another AIF function is to regulate the permeability of the mitochondrial membrane upon apoptosis. Normally it is found behind the outer membrane of the mitochondrion and is therefore secluded from the nucleus. However, when the mitochondrion is damaged, it moves to the cytosol and to the nucleus. Inactivation of AIF leads to resistance of embryonic stem cells to death following the withdrawal of growth factors indicating that it is involved in apoptosis.

Fragmentation describes the process of splitting into several pieces or fragments. In cell biology, fragmentation is useful for a cell during both DNA cloning and apoptosis. DNA cloning is important in asexual reproduction or creation of identical DNA molecules, and can be performed spontaneously by the cell or intentionally by laboratory researchers. Apoptosis is the programmed destruction of cells, and the DNA molecules within them, and is a highly regulated process. These two ways in which fragmentation is used in cellular processes describe normal cellular functions and common laboratory procedures performed with cells. However, problems within a cell can sometimes cause fragmentation that results in irregularities such as red blood cell fragmentation and sperm cell DNA fragmentation.

<span class="mw-page-title-main">DFFA</span> Protein-coding gene in the species Homo sapiens

DNA fragmentation factor subunit alpha (DFFA), also known as Inhibitor of caspase-activated DNase (ICAD), is a protein that in humans is encoded by the DFFA gene.

<span class="mw-page-title-main">DNASE1L3</span> Protein-coding gene in the species Homo sapiens

Deoxyribonuclease gamma is an enzyme that in humans is encoded by the DNASE1L3 gene.

<span class="mw-page-title-main">ENDOG</span> Protein-coding gene in the species Homo sapiens

Endonuclease G, mitochondrial is an enzyme that in humans is encoded by the ENDOG gene. This protein primarily participates in caspase-independent apoptosis via DNA degradation when translocating from the mitochondrion to nucleus under oxidative stress. As a result, EndoG has been implicated in cancer, aging, and neurodegenerative diseases such as Parkinson’s disease (PD). Regulation of its expression levels thus holds potential to treat or ameliorate those conditions.

<span class="mw-page-title-main">Apoptotic DNA fragmentation</span> Cleavage of DNA into tiny pieces during apoptosis

Apoptotic DNA fragmentation is a key feature of apoptosis, a type of programmed cell death. Apoptosis is characterized by the activation of endogenous endonucleases, particularly the caspase-3 activated DNase (CAD), with subsequent cleavage of nuclear DNA into internucleosomal fragments of roughly 180 base pairs (bp) and multiples thereof (360, 540 etc.). The apoptotic DNA fragmentation is being used as a marker of apoptosis and for identification of apoptotic cells either via the DNA laddering assay, the TUNEL assay, or the by detection of cells with fractional DNA content ("sub G1 cells") on DNA content frequency histograms e.g. as in the Nicoletti assay.

Epigenomics is the study of the complete set of epigenetic modifications on the genetic material of a cell, known as the epigenome. The field is analogous to genomics and proteomics, which are the study of the genome and proteome of a cell. Epigenetic modifications are reversible modifications on a cell's DNA or histones that affect gene expression without altering the DNA sequence. Epigenomic maintenance is a continuous process and plays an important role in stability of eukaryotic genomes by taking part in crucial biological mechanisms like DNA repair. Plant flavones are said to be inhibiting epigenomic marks that cause cancers. Two of the most characterized epigenetic modifications are DNA methylation and histone modification. Epigenetic modifications play an important role in gene expression and regulation, and are involved in numerous cellular processes such as in differentiation/development and tumorigenesis. The study of epigenetics on a global level has been made possible only recently through the adaptation of genomic high-throughput assays.

<span class="mw-page-title-main">Chromatin immunoprecipitation</span> Genomic technique

Chromatin immunoprecipitation (ChIP) is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell. It aims to determine whether specific proteins are associated with specific genomic regions, such as transcription factors on promoters or other DNA binding sites, and possibly define cistromes. ChIP also aims to determine the specific location in the genome that various histone modifications are associated with, indicating the target of the histone modifiers. ChIP is crucial for the advancements in the field of epigenomics and learning more about epigenetic phenomena.

<span class="mw-page-title-main">Caspase-activated DNase</span> Protein-coding gene in the species Homo sapiens

Caspase-activated DNase (CAD) or DNA fragmentation factor subunit beta is a protein that in humans is encoded by the DFFB gene. It breaks up the DNA during apoptosis and promotes cell differentiation. It is usually an inactive monomer inhibited by ICAD. This is cleaved before dimerization.

Ischemic cell death, or oncosis, is a form of accidental cell death. The process is characterized by an ATP depletion within the cell leading to impairment of ionic pumps, cell swelling, clearing of the cytosol, dilation of the endoplasmic reticulum and golgi apparatus, mitochondrial condensation, chromatin clumping, and cytoplasmic bleb formation. Oncosis refers to a series of cellular reactions following injury that precedes cell death. The process of oncosis is divided into three stages. First, the cell becomes committed to oncosis as a result of damage incurred to the plasma membrane through toxicity or ischemia, resulting in the leak of ions and water due to ATP depletion. The ionic imbalance that occurs subsequently causes the cell to swell without a concurrent change in membrane permeability to reverse the swelling. In stage two, the reversibility threshold for the cell is passed and the cell becomes committed to cell death. During this stage the membrane becomes abnormally permeable to trypan blue and propidium iodide, indicating membrane compromise. The final stage is cell death and removal of the cell via phagocytosis mediated by an inflammatory response.

FAIRE-Seq is a method in molecular biology used for determining the sequences of DNA regions in the genome associated with regulatory activity. The technique was developed in the laboratory of Jason D. Lieb at the University of North Carolina, Chapel Hill. In contrast to DNase-Seq, the FAIRE-Seq protocol doesn't require the permeabilization of cells or isolation of nuclei, and can analyse any cell type. In a study of seven diverse human cell types, DNase-seq and FAIRE-seq produced strong cross-validation, with each cell type having 1-2% of the human genome as open chromatin.

Pseudoapoptosis can be defined from multiple viewpoints, with an underlying premise of the differences in cellular processes and states relating to apoptosis. Pseudoapoptosis can be referred to as an apoptotic-like cellular state that can be readily reversed, or as a process that induces rapid apoptosis through the introduction of drugs such as bleomycin.

<span class="mw-page-title-main">MNase-seq</span> Sk kasid Youtuber

MNase-seq, short for micrococcal nuclease digestion with deep sequencing, is a molecular biological technique that was first pioneered in 2006 to measure nucleosome occupancy in the C. elegans genome, and was subsequently applied to the human genome in 2008. Though, the term ‘MNase-seq’ had not been coined until a year later, in 2009. Briefly, this technique relies on the use of the non-specific endo-exonuclease micrococcal nuclease, an enzyme derived from the bacteria Staphylococcus aureus, to bind and cleave protein-unbound regions of DNA on chromatin. DNA bound to histones or other chromatin-bound proteins may remain undigested. The uncut DNA is then purified from the proteins and sequenced through one or more of the various Next-Generation sequencing methods.

<span class="mw-page-title-main">Sperm Chromatin Structure Assay</span>

Sperm Chromatin Structure Assay (SCSA) is a diagnostic approach that detects sperm abnormality with a large extent of DNA fragmentation. First described by Evenson in 1980, the assay is a flow cytometric test that detects the vulnerability of sperm DNA to acid-induced denaturation DNA in situ. SCSA measures sperm DNA fragmentation attributed to intrinsic and extrinsic factors and reports the degree of fragmentation in terms of DNA Fragmentation Index (DFI). The use of SCSA expands from evaluation of male infertility and subfertility, toxicology studies and evaluation of quality of laboratory semen samples. Notably, SCSA outcompetes other convention sperm DNA fragmentation (sDF) assays such as TUNEL and COMET in terms of efficiency, objectivity, and repeatability.

References

  1. 1 2 3 Speyer BE, Pizzey AR, Ranieri M, Joshi R, Delhanty JD, Serhal P (May 2010). "Fall in implantation rates following ICSI with sperm with high DNA fragmentation". Hum Reprod. 25 (7): 1609–1618. doi:10.1093/humrep/deq116. PMID   20495207.
  2. Williamson, Robert (1970). "Properties of rapidly labelled deoxyribonucleic acid fragments isolated from the cytoplasm of primary cultures of embryonic mouse liver cells". Journal of Molecular Biology. 51 (1): 157–168. doi:10.1016/0022-2836(70)90277-9. PMID   5481278.
  3. Quail, Michael Andrew (2010). "DNA: Mechanical Breakage". Encyclopedia of Life Sciences. doi:10.1002/9780470015902.a0005333.pub2. ISBN   978-0470016176.
  4. Phillips, Thearesa. "Restriction Enzymes Explained". Biotech / Biomedical. About.com. Archived from the original on 5 June 2016. Retrieved 2 April 2013.
  5. 1 2 3 4 5 6 "DNA Fragmentation". New England Biolabs. Archived from the original on 20 December 2016. Retrieved 2 April 2013.
  6. Sambrook, Joseph; Russell, David W. (2006). "Fragmentation of DNA by Nebulization". Cold Spring Harbor Protocols. Cold Spring Harbor Laboratory Press. 2006 (23): pdb.prot4539. doi:10.1101/pdb.prot4539. PMID   22485920 . Retrieved 3 April 2013.
  7. "Ultrasonic Lysis: Cell Disruption & Extraction Fragmentation" . Retrieved 15 May 2017.
  8. Nagata S (April 2000). "Apoptotic DNA fragmentation". Exp. Cell Res. 256 (1): 12–8. doi:10.1006/excr.2000.4834. PMID   10739646.
  9. Enari, Masato; Sakahira, Hideki; Yokoyama, Hideki; Okawa, Katsuya; Iwamatsu, Akihiro; Nagata Shigekazu (January 1998). "A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD". Nature. 391 (6662): 43–50. Bibcode:1998Natur.391...43E. doi:10.1038/34112. PMID   9422506. S2CID   4407426 . Retrieved 8 April 2013.
  10. Belloc S, Benkhalifa M, Cohen-Bacrie M, Dalleac A, Chahine H, Amar E, Zini A (2014). "Which isolated sperm abnormality is most related to sperm DNA damage in men presenting for infertility evaluation". J. Assist. Reprod. Genet. 31 (5): 527–32. doi:10.1007/s10815-014-0194-3. PMC   4016368 . PMID   24566945.
  11. Simon L, Brunborg G, Stevenson M, Lutton D, McManus J, Lewis SE (May 2010). "Clinical significance of sperm DNA damage in assisted reproduction outcome". Hum Reprod. 25 (7): 1594–1608. doi: 10.1093/humrep/deq103 . PMID   20447937.
  12. Gorczyca W, Traganos F, Jesionowska H, Darzynkiewicz Z (1993). "Presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal human sperm cells. Analogy to apoptosis of somatic cells". Exp Cell Res. 207 (1): 202–205. doi:10.1006/excr.1993.1182. PMID   8391465.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  13. 1 2 Zhang LH, Qiu Y, Wang KH, Wang Q, Tao G, Wang LG (June 2009). "Measurement of sperm DNA fragmentation using bright-field microscopy: comparison between sperm chromatin dispersion test and terminal uridine nick-end labeling assay". Fertil. Steril. 94 (3): 1027–1032. doi: 10.1016/j.fertnstert.2009.04.034 . PMID   19505686.
  14. 1 2 "DNA Forensics". U.S. Department of Energy Genome Programs. Retrieved 8 April 2013.
  15. Gong JP, Traganos F, Darzynkiewicz Z (1994). "A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry". Anal Biochem. 218 (2): 314–319. doi:10.1006/abio.1994.1184. PMID   8074286.{{cite journal}}: CS1 maint: multiple names: authors list (link)