ZOO 405 Week 2 ZOO405byRania Baleelais licensed under aCreative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License This week Compaction levels DNA properties Genome content Lecture notes can be found at
Each of us has enough DNA to reach from here to the sun and back, more than 300 times. DNA levels of compaction, eukaryotes
1st level of compaction: 2nm DNA molecule into 11nm DNA/Nucleosome. 2nd level of compaction: 11nm DNA/Nucleosome into 30nm fiber. 3rd level of compaction: 30nm fiber nto 300nm radial loop domains. 4th level of compaction: 300nm radial loop domains into 700nm chromosomes. From 2nm DNA to 11nm DNA/Nucleosome
1st level of compaction From 2nm DNA to 11nm DNA/Nucleosome DNA/Nucleosome Nucleosome = the repeating structural unit within eukaryotic chromatin. Nucleosome = dsDNA wrapped around an octamer of histone proteins. An octamer is composed 2 copies each of 4 different histones 146bp of DNA make 1.65 turns around the octamer 1st level of compaction 2nm DNA to 11nm DNA/Nucleosome
Vary in length between 20 to 100bp, depending on species and cell type Diameter of the nucleosome - Core DNA = 146 bp This structure shortens the DNA length about 7 fold From 11nm DNA/Nucleosome to 30nm fiber
2nd level of compaction From 11nm DNA/Nucleosome to 30nm fiber 2nd level of compaction 11nm DNA/Nucleosome to 30nm fiber
The nucleosome units are organized into a more compact structure depending on the presence of additional proteins. The linker histone (H1) is a key player: Binds to linker DNA Also binds to nucleosomes This structure shortens the DNA length about another 7 fold (total reduction around 50 fold). 2nd level of compaction 11nm DNA/Nucleosome to 30nm fiber From 30nm fiber to 300nm radial loop domains
3rd level of compaction From 30nm fiber to 300nm radial loop domains Super coiled and relaxed DNA
Topoisomerase has both nicking and ligase activity Non histones proteins Topoisomerases: A number of different types
Form a structural scaffolding to which loops of chromatin are attached forming a nuclear matrix (or chromosome scaffold). Increase or decrease the linking number of a DNA loop by 2 units. Promote chromosome disentanglement e.g. Topoisomerase II (gyrase) & topoisomerase IV 2 groups of topoisomerases based on the number of strands they break
Class I DNA Topoisomerases [I, III and V] Break 1 strand of a DNA helix. ATP independent (except for reverse gyrase) Primarily responsible for relaxing DNA, while reverse gyrase can introduce positive supercoils into DNA. Mechanism involves rotating the broken strand around the intact strand to relax (unwind) the strain on the DNA helix, followed by resealing the ends of the broken strand. Play an important role in DNA replication and transcription (topoisomerase I), and recombination (topoisomerase III). Class I Subclasses: 1)Type IA enzymes: Bacterial topoisomerase I Topoisomerase III Reverse gyrase 2)Type IB enzymes: Eukaryotic and eukaryal viral topoisomerase I Archaeal topoisomerase V Class IISubclasses: 1)Type IIA enzymes: Eukaryotic and eukaryal viral topoisomerase II Gyrase (bacterial topoisomerase II) Topoisomerase IV 2)Type IIB enzymes: Archaeal topoisomerase VI Class II DNA Topoisomerases [II (gyrase), IV and VI]
Break 2 strands of a DNA helix. ATP dependent. Relax DNA (+ve supercoiling) (topoisomerase IV) & introduce ve supercoiling (topoisomerase II). Mechanism involves passing an intact DNA helix through the gap made by the broken DNA helix, then resealing the strands. Play an important role in chromosome condensation (topoisomerase II) and in the segregation of daughter chromosomes during cell division (topoisomerase IV). 3rd level of compaction A 3rd level of compaction involves:
30nm fiber to 300nm radial loop domains A 3rd level of compaction involves: interaction between the 30 nm fiber & the nuclear matrix (filamentous network of proteins in the nucleus= non histone proteins) Leads to the formation of radial loop domains . Topoisomerase II Cleave and seal dsDNA.
Is located in the long axis of the metaphase chromosome. DNA is tightly bound to it at S/MARs locus (scaffold/matrix attachment regions). Radial loop domains Matrix-attachment regions (MAR) or Scaffold-attachment regions (SARs) SARs are anchored to the nuclear matrix, thus creating radial loops 25000 to bp Histone-depleted metaphase chromosome
Scaffold/Matrix attachment regions Human mitotic chromosomes
DNA is stained blue, and the axis is stainedred(fluorescent antibody against aproteinin the condensincomplex). Only part of the scaffolding is visible. (A) A typicalmitotic chromosome, with a gently coiled scaffold along each of the twochromatids. (B) Ametaphasechromosome, the scaffold has condensed by further helical folding. (Courtesy of Ulrich Laemmli and Kazuhiro Maeshima) 3rd level of compaction The attachment of radial loops to the nuclear matrix is important in 2 ways: 1.It serves to organize the chromosomes within the nucleus: Each chromosome in the nucleus is located in a discrete & non-overlapping chromosome territory. 2.It plays a role in gene regulation. From 300nm radial loop domains to 700nm chromosomes
4th level of compaction From 300nm radial loop domainsto 700nm chromosomes 4th level of compaction 4th level of compaction From 300nm radial loop domains to 700nm chromosomes The compaction level of chromosomes is not completely uniform: Euchromatin: Less condensed regions of chromosomes Transcriptionally active Regions where 30 nm fiber forms radial loop domains Heterochromatin: Tightly compacted regions of chromosomes Transcriptionally inactive (in general) Radial loop domains compacted even further Different forms of chromatin show differential gene activity
Heterochromatin Euchromatin Special cases Sperm heads
DNA must be further compressed. Protamines (mostly Arg) are shuttled to nucleus of developing spermatocytes, where they displace histones. Greater degree of compaction associated with: Maximum compaction= No need for gene expression. Built for speed DNA properties 1. Base pairing 2. Grooves 3. Sense and antisense
4. Supercoiling 5. Alternate DNA structures 6. Quadruplex structures 7. Branched DNA 1. Complementary base pairing
DNA double strands are held together due to the complementary base pairing as follows: A =T (automatically form 2 hydrogen bonds). G C (automatically form 3 hydrogen bonds). DNA stability DNA with high GC-content is more stable than that with low GC-content. DNA stability is a result of: hydrogen bonding+GC% +overall length of a DNA double helix. 2. Grooves: provide binding sites
The grooves are unequally sized: The major groove (22 wide)=> edges of the bases are more accessible=> proteins (e.g. transcription factors) contacts the exposed sides of bases in the major groove. The minor groove (12 wide). As the strands are not directly opposite each other, t 3. Sense & antisense DNA Sense (+ve) DNA sequence= runs from 5 to 3 end= coding strand= has the same sequence as the mRNA Antisense (-ve)= mRNA template= compliment. Both sense and antisense sequences can exist on different parts of the same DNA. DNA normally has two strands, i.e., the sense strand and the antisense strand. In double-stranded DNA, only one strand codes for the RNA that is translated into protein. This DNA strand is referred to as the antisense strand. The strand that does not code for RNA is called the sense strand. Proposed functions of antisense: Antisense RNAs are involved in regulating gene expression through RNA-RNA base pairing? A few DNA sequences in prokaryotes and eukaryotes, and more in plasmids and viruses, blur the distinction between sense and antisense strands by having overlapping genes. Overlapping genes, may do double duty, encoding one protein when read along one strand, and a second protein when read in the opposite direction along the other strand. Overlapping genes in bacteria may be involved in the regulation of gene transcription, while in viruses, overlapping genes increase the amount of information that can be encoded within the small viral genome. The two complementary strands of double-stranded DNA (dsDNA) are usually differentiated as the "sense" strand and the "antisense" strand. The DNA sense strand looks like themessenger RNA(mRNA) and can be used to read the expected protein code by human eyes (e.g. ATG codon = Methionine amino acid). However, the DNA sense strand itself is not used to make protein by the cell. It is the DNA antisense strand which serves as the source for the protein code, because, with bases complementary to the DNA sense strand, it is used as a template for the mRNA. Since transcription results in an RNA product complementary to the DNA template strand, the mRNA is complementary to the DNA antisense strand. The mRNA is what is used for translation (protein synthesis). 4. Supercoiling DNA can be twisted like a rope=>DNA supercoiling.
With DNA in its "relaxed" state, a strand usually circles the axis of the double helix once every 10.4 bp. DNA is twisted in the direction of the helix=> positive supercoiling, and the bases are held more tightly together. DNA twisted in the opposite direction=> negative supercoiling, and the bases come apart more easily. Supercoiling in nature
Most DNA has slight -ve supercoiling that is introduced by topoisomerases. Topoisomerases are needed to relieve the twisting stresses introduced into DNA strands during processes such as transcription and DNA replication. 5. Alternate DNA structures
DNA conformations : A-DNA form B-DNA form Z-DNA form. Only B-DNA & Z-DNA were observed in functional organisms. A, B & Z-DNA Backbone A-T bp G-C bp Z-DNA B-DNA A-DNA DNA conformation depends on
The hydration level, DNA sequence, The amount and direction of supercoiling, Chemical modifications of the bases, The type and concentration of metal ions, The presence of polyamines in solution. The conformation that DNA adopts depends on: B-DNA form Is the most common, Occur at high hydration levels,
Is not a well-defined conformation but a family of related DNA conformations. A- DNA Occurs under non-physiological conditions in partially dehydrated samples of DNA, In the cell it may be produced in hybrid pairings of DNA and RNA strands, as well as in enzyme-DNA complexes. The A-DNA form is a wider right-handed spiral, with a shallow, wide minor groove and a narrower, deeper major groove. Z-DNA Segments of DNA where the bases have been chemically modified by methylation may undergo a larger change in conformation and adopt the Z form (the strands turn about the helical axis in a left-handed spiral. Can be recognized by specific Z-DNA binding proteins and may be involved in the regulation of transcription. 6. Quadruplex structures
Nucleic acid sequences, rich in guanine, are capable of forming four-stranded structures => G-quadruplexes (G-tetrads or G4-DNA). G-quadruplexes may stabilize chromosome ends (telomeres). Potential quadruplex sequences have been identified in G-rich eukaryotic telomeres, and more recently in non-telomeric genomic DNA, e.g. in nuclease-hypersensitive promoter regions Stabilized by Hydrogen bonds between the edges of the bases,
Chelation of a metal ion in the centre of each 4-base unit. Interesting as targets for therapeutic intervention Some sequences in cancer-related genes have been identified as forming quadruplex structures E.g. DNA quadruplex formed by telomere repeats.
4 G bases form a flat plate and these flat 4-base units then stack on top of each other, to form a stable G-quadruplex structure 7. Branched DNA Branched DNA occurs: If a third strand of DNA is introduced & contains adjoining regions able to hybridize with the frayed regions of the pre-existing double-strand. Single branch Multiple branches Branched DNA: application in nanotechnology
Nanotechnology is the engineering of functional systems at the molecular scale. DNA is used as a structural material rather than as a carrier of genetic information. Possible applications in molecular self-assembly and in DNAcomputing. DNA-nanotechnology Nadrian Seeman was inspired by the woodcut Depth to realize that a 3D DNA lattice could be used to orient target molecules, simplifying their crystallographic study. Depth A woodcut by M. C. Escher Nadrian Seeman A crystallographer, NY Uni.
In 1991, Seeman's laboratory synthesized a DNA cube followed by a DNA truncated octahedron (cons: not rigid enough to form 3D lattices). Seeman developed the more rigid double-crossover (DX) motif (tile-based structures; can be used for DNA computing). In 2009, Seeman published the synthesis of a 3D DNA lattice. 4-arm junction 4 DNA single strands This work inspired the concept of DNA-nanotech A double-crossover (DX) molecule 5 DNA single strands Molecular diagnostic assays using bDNA technology for detection of NA target molecules
Are sensitive, specific and reliable tools in the diagnosis of viral and bacterial infections and for monitoring disease progression during the course of therapy. bDNA tests are less labour-intensive than many molecular-based procedures. Using bDNA, amplification of a target sequence is not required, and, thus, cross-contamination between replicate samples due to excessive amplicons or carryover is less likely in bDNA assays. Developed for the detection of: Trypanosoma brucei, antibiotic-sensitive and antibiotic-resistant Staphylococcus, papillomavirus and hepatitis B virus Steps in a bDNA assay for viral load
Used in the US for quantification of HIV and Hepatitis C load in patients 1. Capture stage: disruption of organism
Ensure that viral particles have been disrupted and that viral RNA is present for analysis 2. bDNA target capture Target-specific oligonucleotides (label extenders and capture extenders) then are hybridized with high stringency to the target nucleic acid 3. bDNA signal amplification
Capture extenders are designed to hybridize to the target and to capture probes, which are attached to a microwell plate. Label extenders are designed to hybridize to contiguous regions on the target and to provide sequences for hybridization of a preamplifier oligonucleotide. Signal amplification begins with preamplifier probes hybridizing to label extenders. Other regions on the preamplifier are designed to hybridize to multiple bDNA amplifier molecules that create a branched structure 4. bDNA detection Alkalinephosphatase (AP)-labeled oligonucleotides, which are complementary to bDNA amplifier sequences, hybridize with the bDNA molecule. The bDNA signal is the chemi-luminescent product of the AP reaction= proportional to the number of AP-labeled probes that hybridize to bDNA secondary sequences Absolute quantification is accomplished by establishing a STD curve for each run Bayer System 340 bDNA Analyzer