User:Dbrook22/sandbox

Article Evaluation

Article chosen: CpG site

Everything in the article appears related to the topic of CpG sites and offers interesting links of CpG site methylations to genetic diseases like cancer.

The article appears to be neutral with only fact-based statements presented and no personal opinions offered.

All viewpoints seem to be given equal weight.

There was a reference made to the point of 70-80% abundance of methylated CpG sites, yet the source referred to doesn't have this data. The author may have gotten this reference confused with another. A suggestion to change this has been made.

All the links tested are functional.

The references made are to reputable sources such as the Journal of Molecular Biology and The American Journal of Pathology.

A few of the sources used are not recent. One being a study on sea urchin embryos published in 1967.

A suggestion that I would make is in terms of the sub-headings. The introductory section seems to try and cover too much initially which causes an overwhelming amount of information delivered right away. For example, frequency and detection of methylated CpG sites can each be divided into their own sub-sections, considering they are important details.

As well as making detection of CpG sites it's own subsection, more information should be provided about this. The article lacks the description of many genetic technologies that we have already touched on in 3595 class. To make this a more up-to-date and relevant article, innovative technologies should be described in relation to the topic of CpG sites and their methylation.

On the Talk page, there is critique about the image shown of CpG sites highlighted among a sea of genetic code. The yellow font of this graphic makes it very difficult to visualize on the white background, thus weakening the strength of the image. It was suggested to change this colour so that one can more easily observe these CpG sites in the genetic code.

The article is rated a C with a Mid-range importance. It is currently part of a Wikiproject on Genetics/Statistics.

This article discusses CpG sites in a similar way to 3595 class. Something it doesn't touch on as much as us is the links of CpG site methylation to diseases other than cancer. As we've seen already, CpG methylated sites can lead to diseases like Fetal Alcohol Spectrum Disorder (FASD). A more diverse explanation of the other resulting diseases would be needed.

Week 5- Possible Articles

DNA unwinding element- More information required about how it works, homology to other proteins, who discovered it, and structure.

Genetic imbalance- Good information so far about its effects, but information needed regarding how it arises and ways to prevent this imbalance.

Histone 3' UTR stem-loop- Very good introduction about what it is. Supplementary information needed.

Methylation specific oligonucleotide microarray- Barely a stub so far. It will be needed to edited basically from scratch. Description of function, its uses, the history of its development, pictures, and more.

Looks good so far. Keep it up! AdamCF87 (talk) 15:13, 17 October 2017 (UTC)

Week 7- Finding Sources + Information

Possible Sources

The DNA unwinding element: a novel, cis-acting component that facilitates opening of the Escherichia coli replication origin.

• Origin caused by unstable helices within the DNA unwinding element via supercoiling
• Vital for access of replication machinery to undergo DNA replication
• Supercoiling --> Unstable helix --> Ease of unwinding

Structure and Function of the c-myc DNA-unwinding Element-binding Protein DUE-B

• Prokaryotes and eukaryotes
• Replication origins
• DNA unwinding element proteins bind at these sites
• Easily unwound in nature
• (DUE)
• Essential for chromosomal replication origin activity
• Deletion of the DUE region = no more origin activity
• Can be restored by re-addition of DUE site
• Has proteins that bind to it (DUE-B) that help initiate replication
• DUE-B's found in eukaryotes, bacteria, yeast
• DUE-B homologs in fish, rodents, amphibians
• HeLa cells- knock down of DUE-B --> no entry into S phase + apoptosis
• Interspecies conservation: Knockout of DUE-B in Xenopus egg extracts = no DNA replication. Activity regained by addition of HeLa DUE-B
• DUE-B homodimerizes --> forms continuous beta-sheet structure between 2 monomers, extending across the homodimer interface. Asymmetrical unit containing 4 copies of the monomer = 2 copies of the DUE-B homodimer in the total asymmetrical unit
• DUE-B C-terminal regions that interact with the DUE DNA are highly disordered- nucleotide binding motif. Region not involved in dimerization (experiment: truncated this region and dimerization still occurred). C terminus removed by proteolysis
• DUE-B: ATPase and D-aminoacyl-tRNA deacylase activity
• DUE-B: 209 residues, 58 unstructured when ligand-free
• DUE-B exact crystal structure given
• DUE-B homodimers and heterodimers formed in vivo are stable over the cell cycle
• Disordered regions become ordered upon binding to the DUE--> full length protein resistant to protease digestion vs. not bound to DNA = significantly less resistant
• DUE-B binds c-myc DUE in vitro
• Double stranded DNA protects the C terminus of DUE-B against proteases
• C terminus is necessary for binding of DUE-B to DNA- not other sequence specificity

Eukaryotic DNA Replication: Anatomy of An Origin

• Prokaryotic replication origins require: DUE, recognition sequence, and binding sites for transcription factors
• Rate may be impeded by chromatin structure
• Linear genes- require less energy to unwind DNA
• Site where DNA unwinding begins
• Three required domains (as seen with 64-bp SV40 ori core analysis): 1) 17-bp region with one T-rich strand and one A-rich strand, 2) 24-bp region with 4 symmetrically placed pentamer binding sites for T-ag, 3) 10-bp sequence in the early palindrome
• Thermoanalysis of this SV40 ori: Nucleotides 5209-5217 as DUE region
• DUE in yeast: Lies 3' to the T-rich strand of ori core. Organization of T-ag hexamers required for unwinding- covers DUE and initiates melting of DNA strands --> single-stranded
• T-ag hexamers covering A/T-domains initiates a compensating twist to the DUE of ori core
• Ori core consists of: Origin Recognition Element (ORE), DNA-unwinding Element (DUE), and A/T-rich element in which one strand is A-rich and one is T-rich- spacing, orientation, and arrangement of these three is vital to ori function
• Sometimes that A/T-rich element serves as the DUE itself- like in HSV
• Easily unwound DNA region
• Not a unique sequence
• Determined by base-stacking interactions
• Dependent on nucleotide sequence AND AT content
• Insensitive to point mutations-- if alter close matches to protein binding sites but don't reduce ARS (autonomously-replicating sequences) activity, do not reduce DUE activity
• Sensitive to large sequence changes
• Sensitive to reactive reagents and ss-specific-nucleases
• First demonstrated by Kowalski- site where DNA synthesis begins. In yeast and bacteria origins at first
• Yeast: DUE lies within ~100 bp flanking 3'-end of T-rich strand of the ORE-- mutations here inhibit origin activity in vivo. Effect of this mutation can be partially reversed by elevated temperature
• 3 cis-acting elements (B1, B2, B3) that also effect ARS (autonomously replicating sequences) activity. B2 functions as a DUE
• ABF1 (transcription factor) can modulate DUE activity

Nucleic Acids Research

• Unaffected by easily unwound sequences present elsewhere on same plasmid
• Entry site for replicative enzymes into DNA helix
• Mutations decreasing occurrence of unwinding at DUE decrease replication activity
• Substrate for the unwinding activity of the replication initiation complex
• Properly positioned in relation to an initiator protein binding site

Base Pair Opening in Three DNA-unwinding Elements

• Specific base sequences
• Located in the origin of DNA replication
• Start point for DNA helix unwinding/ strand separation
• Correlation found between kinetics and energetics of opening of AT/TA base pairs and the location of the corresponding DNA-unwinding element in the origin of DNA replication
• Unwinding even occurs in absence of origin-binding proteins. Induced by supercoiling alone.
• Molecular origin is not yet understood
• Analysis on free energy has been made
• Properties examined of three bacterial DUEs using imino exchange and NMR spectroscopy. Characterization of the stability and dynamics of the three DNA double helices at the level of individual base pairs

The c-myc DNA-unwinding Element-binding Protein Modulates the Assembly of DNA Replication Complexes in Vitro

• Eukaryotes-
  • Do they contribute to origin activity by their intrinsic helical instability, as protein-binding sites, or both?

• Humans-
  • Based on homology to yeast proteins, previously classified as an aminoacyl-tRNA synthetase
  • ~60 amino acids longer than orthologs in yeast and worms
  • Primarily nuclear

• DUE-B levels constant during the cell cycle, although the protein is preferentially phosphorylated in cells arrested early in S phase
• Inhibition of DUE-B protein expression slowed HeLa cell cycle progression from G1 to S phase and induced cell death
• In Xenopus extracts, baculovirus-expressed DUE-B inhibited chromatin replication and replication protein A loading in the presence of endogenous DUE-B, suggesting that differential covalent modification of these protein can alter their effect on replication
• Recombinant DUE-B expressed in HeLa cells restored replication activity to egg extracts immunodepleted with anti-DUE-B antibody, suggesting that DUE-B plays an important role in replication in vivo
• The initiation of DNA replication in eukaryotes relies on the sequential assembly of protein complexes at replicator sequences, controlled by the activities of kinases and phosphatases
• In S. cerevisiae, chromosomal replication origins cloned in plasmids display autonomously replicating sequence (ARS) activity characteristically comprise a set of modular elements, including an ARS consensus sequence (ACS) (4)-binding site for ORC, a region of helical instability termed a DNA-unwinding element (DUE) that contributes to origin activity through template template unwinding or binding of pre-RC proteins, and transcription factor-binding sites that can promote the assembly of replication complexes through protein-protein interactions and modification of chromatin structure
• In mammalian chromosomes, no consensus DNA sequence analogous to the yeast initiator-binding site has been identified. Instead, the feature most common to mammalian origins is a region of helical instability
  • Ectopic assays of β-globin, lamin B2, and c-myc loci reveal the minimal elements essential for replication
  • c-myc origin core satisfies the genetic criteria of a chromosomal replicator and that a short segment of the c-myc replicator containing the DUE and three matches to the yeast ACS is essential for replicator activity
  • Use of a yeast one-hybrid assay to isolate proteins that bind to the c-myc DUE/ACS region
    • Found one- DUE-B
      • Predicted molecular mass of 23.4 kDA
      • Strong evolutionary conservation in yeast, mice, frogs, and flies
• Chromatin immunoprecipitation assays also show that DUE-B bound at or near the c-myc replicator DUE in a cell cycle-dependent manner in vivo
• SV40- a viral genome
• HeLa cells-
  • DUE-B is a constitutively expressed protein found attached to chromatin and in the soluble fraction of lysed cells
  • ~46 kDa measured by gel exclusion chromatography
  • Co-purified with ATPase activity
  • Down-regulation of DUE-B protein expression by small interfering RNA (siRNA) was associated with a prolonged G1 phase and the induction of cell death
• Main finding: c-myc DUE-B plays a role in regulating replication initiation in HeLa cells
• Deletion or substitution of the c-myc DUE/ACS region strongly suppresses chromosomal replicator activity
  • Isogenic strains with mutations in DUE did not have its cDNA promote rapid colony growth under selective conditions in the same way that the wild type did
• Yeast proteins interacting with the c-myc ACS may block access of the Gal4AD-DUE-B fusion protein or other transcription factors to the reporter
• DUE-B: open reading frame of 209 amino acids
  • Provisionally annotates as a human histidyl-aminoacyl-tRNA synthetase (HARS2). Not validated though
• Human DUE-B has notable similarity to: Mus musculus (98%) and X.laevis (89%)
• DUE-B cDNA spans seven exons on human chromosome 20, with the proposed initiator methionine located in exon 2
  • Northern blot analysis revealed a single species of ~1.4-kb DUE-B mRNA, sufficient to encode a 209 amino acid long protein
• The C-terminal 60-amino acid extension of the human protein ot found in the worm and yeast enzymes is conserved in the frog (70% identical) and mouse (93% identical) proteins
• DUE-B found to play a role in modulating DNA replication in vivo
• Over its N-terminal 148 amino acids, DUE-B shows strong similarity (>45% identical and >65% homologous) to yeast and bacterial homodimeric D-tyrosyl-tRNA^Tyr deacylases
  • HeLa DUE-B bound to chromatin: Its depletion is associated with decreased cell proliferation
  • Vs., DUE-B ins S. cerevisiae which is nonessential and cytoplasmic
    • Thus, the yeast and human enzymes appear to have some dissimilar properties
• DUE-B appears to be present at roughly constant levels throughout the cell cycle and agents that inhibit replication (e.g., aphidicolin and hydroxyurea) did not affect the levels or cellular distribution of DUE-B.
  • DUE-B does not appear to redistribute between cellular compartments in response to DNA damage
• Observation; DUE-B showed preferential binding to the wild-type DUE/ACS sequence in vivo and in vitro in the presence of HeLa nuclear protein suggests that DUE-B binding to the DUE/ACS may be indirect or involved other proteins that influence the structure of DNA
• DUE-B and the MCM helicase recognize/ modulate the structure of the DUE
• The idea that the binding of DUE-B to the DUE is affected by the proximal binding of other proteins is consistent with the enhanced expression of the one-hybrid reporter in yeast when the ARS consensus elements flanking the c-myc DUE were mutated
• Upon gel exclusion chromatography, DUE-B expressed in bacteria eluted at a position consistent with its predicted monomeric molecular mass, whereas DUE-B expressed from a baculovirus vector eluted as a dimer, suggesting that expression in the eukaryotic cells allows post-translational modifications that affect DUE-B function
  • High molecular mass complexes containing DUE-B may bind more weakly to chromatin than the dimeric form
• Relation to disease:
  • Preliminary results found indicating that DUE-B mRNA levels were elevated by 40-300% in 15 of 20 ovarian and colon tumours tested relative to neighbouring normal tissue

DNA helical stability accounts for mutational defects in a yeast replication origin

• DUE- required sequence that is hypersensitive to single-strand-specific nucleases
  • Serves to facilitate origin unwinding
• DUE can be identified in the C2G1 ARS, a chromosomal replication origin, by using a computer program that calculates DNA helical stability from the base sequence
• Nucleotide-level mapping shows that the nuclease-hypersensitive site at the ARS spans a 100-base-pair sequence in the required 3'-flanking region
  • Mutations that stabilize the DNA helix in the broad 3'-flanking region reduce or abolish ARS-mediated plasmid replication, indicating that helical instability is required for origin function
  • The level of helical instability is quantitively related to the replication efficiency of the ARS mutants
• DUE is a conserved component of the C2G1 ARS and is a major determinant of replication origin activity
• DUE: Conserved component of DNA replication origins in cells of bacteria, yeast, and mammals
• Hyper-sensitive to single-strand-specific nucleases
  • Suprisingly not due to high A-T content- also doesn't effect replication origin function
  • Largely determined by stacking interactions between nearest-neighbor bases and, therefore, depends on the DNA sequence
• Low free-energy requirement for localized DNA unwinding
• Helical stability at the 3'-flanking region can alone account for the deleterious effects of mutations on the replication efficiency of the C2G1 ARS

DNA unwinding element

DNA unwinding at the DUE, allowing for formation of replication fork for DNA replication to occur.

From Wikipedia, the free encyclopedia

A DNA unwinding element', frequently abbreviated to DUE and on occasion DNAUE, is the initiation site for the opening of the double helix structure of the DNA at the origin of replication for DNA synthesis. It is A-T rich and denatures easily due to its low helical stability, which allows the single-strand region to be recognized by Origin Recognition Complex.

DUEs are found in both prokaryotic and eukaryotic organisms, but were first discovered in yeast and bacteria origins, by Huang Kowalski . The DNA unwinding allows for access of replication machinery to the newly single strands. In eukaryotes, DUEs are the binding site for DNA-unwinding element binding (DUE-B) proteins required for replication initiation. In prokaryotes, DUEs are found in the form of tandem consensus sequences flanking the 5' end of DnaA binding domain. The act of unwinding at this A-T rich element though, occurs even in absence of any origin binding proteins due to negative supercoiling forces, making it an energetically favourable action. DUEs are typically found spanning 30-100 bp of replication origins.

Process

The specific unwinding of the DUE allows for initiation complex assembly at the site of replication on single-stranded DNA. The DNA helicase and associated enzymes are now able to bind to the unwound region, creating a replication fork start.^[8] The unwinding of this duplex strand region is associated with a low free energy requirement, due to helical instability caused by specific base-stacking interactions, in combination with counteracting supercoiling. Negative supercoiling allows the DNA to be stable upon melting, driven by reduction of torsional stress. Found in the replication origins of both bacteria and yeast, as well as present in some mammalian ones. Found to be between 30-100 bp long.

Prokaryotes

In prokaryotes, most of the time DNA replication is occurring from one single replication origin on one single strand of DNA sequence. Whether this genome is linear or circularized, bacteria have own machinery for replication to occur.

Process

In bacteria, the protein DnaA is the replication initiator. It gets loaded onto oriC at a DnaA box sequence where binds and assemble filaments to open duplex and recruit DnaB helicase with the help of DnaC. DnaA is highly conserved and has two DNA binding domains. Just upstream to this DnaA box, is three tandem 13-mer sequences. These tandem sequences, labelled L, M, R from 5' to 3' are the bacterial DNA unwinding elements. Two out of three of these A-T rich regions (M and R) become unwound upon binding of DnaA to DnaA box, via close proximity to unwinding duplex. The final 13-mer sequence L, farthest from this DnaA box eventually gets unwound upon DnaB helicase encircling it. This forms a replication bubble for DNA replication to then proceed.

Favourability

Unwinding of these three DUEs is a necessary step for DNA replication to initiate. The distant pull from duplex melting at the DnaA box sequence is what induces further melting at the M and R DUE sites. The more distant L site is then unwound by DnaB binding. Unwinding of these 13-mer sites is independent of oriC-binding proteins. It is the generation of negative supercoiling that causes the unwinding.

The rates of DNA unwinding in the three E.coli DUEs were experimentally compared through nuclear resonance spectroscopy. In physiological conditions, the opening efficiency of each of the A-T rich sequences differed from one another. Largely due to the different distantly surrounding sequences.

Additionally, melting of AT/TA base pairs were found to be much faster than that of GC/CG pairs (15-240s^-1 vs. ~20s^-1). This supports the idea that A-T sequences are evolutionarily favoured in DUE elements due to their ease of unwinding.

Consensus Sequence

The three 13-mer sequences identified as DUEs in E.coli, are well-conserved at the origin of replication of all documented intestinal bacteria. A general consensus sequence was made via comparison of conserved bacteria to form an 11 base sequence. E.coli contains 9 bases of the 11 base consensus sequence in its oriC, within the 13-mer sequences. These sequences are found exclusively at the origin of replication; not anywhere else within the genome sequence.

Eukaryotes

Eukaryotic replication mechanisms work in relatively similar ways to that of prokaryotes, but is under much more finely-tuned regulation. There is a need to ensure that each DNA molecule is replicated only once and that this is occurring in the proper location at the proper time. Operates in response to extracellular signals that coordinate initiation of division, differently from tissue to tissue. External signals trigger replication in S phase via production of cyclins which activate cyclin-dependent kinases (CDK) to form complexes.

DNA replication in eukaryotes initiates upon origin recognition complex (ORC) binding to the origin. This occurs at G₁ cell phase serving to drive the cell cycle forward into S phase. This binding allows for further factor binding to create a pre-replicative complex (pre-RC). Pre-RC triggered to initiate when cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) bind to it. Initiation complexes then allow for recruitment for helicase and unwinding of duplex at origin.

Replication in eukaryotes is initiated at multiple sites on the sequence, forming multiple replication forks simultaneously. This efficiency is required with the large genomes that they need to replicate.

Yeast

Studies in yeast have localized these DUE regions 3’ to autonomously replicating sequence (ARS). Differences in DUE sequence have impact on the efficiency of these ARS on replication initiation, results gained from imino exchange and NMR spectroscopy. Have been found to span approximately 100 bases in region of origin initiation.

Mammals

In eukaryotes, nucleosome structures can complicate replication initiation. They can block access of DUE-B’s to the DUE, thus suppressing transcription initiation. Can impede on rate. The linear nature of eukaryotic DNA, vs prokaryotic circular DNA, though, is easier to unwind its duplex once has been properly unwound from nucleosome. In eukaryotes, activity of DUE can be modulated by transcription factors like ABF1.

DUE in origin of plasmids in mammalian cells, SV40, found to be associated with a T-ag hexamer, that introduces opposite supercoiling to increase favourabilty of strand unwinding.

Mammals- Many contain DUE that forms cruciforms, intramolecular triplexes, or other alternative DNA structures that may present DNA in a single-strand form. Where replication complexes assemble. Chromosomes of higher eukaryotes have defined replication origins, but few replication origins have been characterized in human cells. Initiation begins in broad region called initiation zone. In humans, characterized at origins associated with several genes, including c-myc gene and the beta-globin gene.

DUE-binding proteins (DUE-Bs)

DNA unwinding element proteins (DUE-B) are found in eukaryotes.

They act to initiate strand separation by binding to DUE. DUE-B sequence homologs found among a variety of animal species- fish, amphibians, and rodents. DUE-B’s have disordered C-terminal domains that bind to the DUE by recognition of this C-terminus. No other sequence specificity involved in this interaction. Confirmed by inducing mutations along length of DUE-B sequence, but in all cases dimerization abilities remaining intact. Upon binding DNA, C-terminus becomes ordered, imparting a greater stability against protease degradation. DUE-B’s are 209 residues in total, 58 of which are disordered until bound to DUE. DUE-B’s hydrolyze ATP In order to function. Also possess similar sequence to aminoacyl-tRNA synthetase, and were previously classified a such. DUE-Bs form homodimers that create an extended beta-sheet secondary structure extending across it. Two of these homodimers come together to form the overall asymmetric DUE-B structure.

In humans, DUE-B’s are 60 amino acids longer than its yeast ortholog counterparts. Both localized mainly in the nucleus.

DUE-B levels are in consistent quantity, regardless of cell cycle. In S phase though, DUE-Bs can be temporarily phosphorylated to prevent premature replication. DUE-B activity is covalently controlled. The assembly of these DUE-Bs at the DUE regions is dependent on local kinase and phosphatase activity. DUE-B’s can also be down-regulated by siRNAs and have been implicated in extended G1 stages.

Associated Problems/ Mutations

Mutations that impair the unwinding at DUE sites directly impede DNA replication activity. This can be a result of deletions/changes in the DUE region, the addition of reactive reagents, or the addition of specific nuclease. DUE sites are relatively insensitive to point mutations though, maintaining their activity in when altering bases in protein binding sites. In many cases, DUE activity can be partially regained by increasing temperature. Can be regained by the re-addition of DUE site as well.

In eukaryotes, when DUE-B's are knocked out, the cell will not go into S phase of its cycle, where DNA replication occurs. Increased apoptosis will result. But, activity can be rescued by re-addition of the DUE-B's, even from a different species. This is because DUE-B's are homologous between species. For example, if DUE-B in Xenopus egg are mutated, no DNA replication will occur, but can be saved by addition of HeLa DUE-B's to regain full functionality.