DNA-binding proteins

DNA-binding proteins

Interaction of DNA with histones (shown in white, top). These proteins' basic amino acids (below left, blue) bind to the acidic phosphate groups on DNA (below right, red).

Structural proteins that bind DNA are well-understood examples of non-specific DNA-protein interactions. Within chromosomes, DNA is held in complexes with structural proteins. These proteins organize the DNA into a compact structure called chromatin. In eukaryotes this structure involves DNA binding to a complex of small basic proteins called histones, while in prokaryotes multiple types of proteins are involved.^[63][64] The histones form a disk-shaped complex called a nucleosome, which contains two complete turns of double-stranded DNA wrapped around its surface. These non-specific interactions are formed through basic residues in the histones making ionic bonds to the acidic sugar-phosphate backbone of the DNA, and are therefore largely independent of the base sequence.^[65] Chemical modifications of these basic amino acid residues include methylation, phosphorylation and acetylation.^[66] These chemical changes alter the strength of the interaction between the DNA and the histones, making the DNA more or less accessible to transcription factors and changing the rate of transcription.^[67] Other non-specific DNA-binding proteins found in chromatin include the high-mobility group proteins, which bind preferentially to bent or distorted DNA.^[68] These proteins are important in bending arrays of nucleosomes and arranging them into more complex chromatin structures.^[69]

A distinct group of DNA-binding proteins are the single-stranded-DNA-binding proteins that specifically bind single-stranded DNA. In humans, replication protein A is the best-characterised member of this family and is essential for most processes where the double helix is separated, including DNA replication, recombination and DNA repair.^[70] These binding proteins seem to stabilize single-stranded DNA and protect it from forming stem-loops or being degraded by nucleases.

The lambda repressor helix-turn-helix transcription factor bound to its DNA target^[71]

In contrast, other proteins have evolved to specifically bind particular DNA sequences. The most intensively studied of these are the various classes of transcription factors, which are proteins that regulate transcription. Each one of these proteins bind to one particular set of DNA sequences and thereby activates or inhibits the transcription of genes with these sequences close to their promoters. The transcription factors do this in two ways. Firstly, they can bind the RNA polymerase responsible for transcription, either directly or through other mediator proteins; this locates the polymerase at the promoter and allows it to begin transcription.^[72] Alternatively, transcription factors can bind enzymes that modify the histones at the promoter; this will change the accessibility of the DNA template to the polymerase.^[73]

As these DNA targets can occur throughout an organism's genome, changes in the activity of one type of transcription factor can affect thousands of genes.^[74] Consequently, these proteins are often the targets of the signal transduction processes that mediate responses to environmental changes or cellular differentiation and development. The specificity of these transcription factors' interactions with DNA come from the proteins making multiple contacts to the edges of the DNA bases, allowing them to "read" the DNA sequence. Most of these base-interactions are made in the major groove, where the bases are most accessible.^[75]

The restriction enzyme EcoRV (green) in a complex with its substrate DNA^[76]

DNA-modifying enzymes

Nucleases and ligases

Nucleases are enzymes that cut DNA strands by catalyzing the hydrolysis of the phosphodiester bonds. Nucleases that hydrolyse nucleotides from the ends of DNA strands are called exonucleases, while endonucleases cut within strands. The most frequently-used nucleases in molecular biology are the restriction endonucleases, which cut DNA at specific sequences. For instance, the EcoRV enzyme shown to the left recognizes the 6-base sequence 5′-GAT|ATC-3′ and makes a cut at the vertical line. In nature, these enzymes protect bacteria against phage infection by digesting the phage DNA when it enters the bacterial cell, acting as part of the restriction modification system.^[77] In technology, these sequence-specific nucleases are used in molecular cloning and DNA fingerprinting.

Enzymes called DNA ligases can rejoin cut or broken DNA strands.^[78] Ligases are particularly important in lagging strand DNA replication, as they join together the short segments of DNA produced at the replication fork into a complete copy of the DNA template. They are also used in DNA repair and genetic recombination.^[78]

Topoisomerases and helicases

Topoisomerases are enzymes with both nuclease and ligase activity. These proteins change the amount of supercoiling in DNA. Some of these enzyme work by cutting the DNA helix and allowing one section to rotate, thereby reducing its level of supercoiling; the enzyme then seals the DNA break.^[23] Other types of these enzymes are capable of cutting one DNA helix and then passing a second strand of DNA through this break, before rejoining the helix.^[79] Topoisomerases are required for many processes involving DNA, such as DNA replication and transcription.^[24]

Helicases are proteins that are a type of molecular motor. They use the chemical energy in nucleoside triphosphates, predominantly ATP, to break hydrogen bonds between bases and unwind the DNA double helix into single strands.^[80] These enzymes are essential for most processes where enzymes need to access the DNA bases.

Polymerases

Polymerases are enzymes that synthesize polynucleotide chains from nucleoside triphosphates. The sequence of their products are copies of existing polynucleotide chains - which are called templates. These enzymes function by adding nucleotides onto the 3′ hydroxyl group of the previous nucleotide in a DNA strand. Consequently, all polymerases work in a 5′ to 3′ direction.^[81] In the active site of these enzymes, the incoming nucleoside triphosphate base-pairs to the template: this allows polymerases to accurately synthesize the complementary strand of their template. Polymerases are classified according to the type of template that they use.

In DNA replication, a DNA-dependent DNA polymerase makes a DNA copy of a DNA sequence. Accuracy is vital in this process, so many of these polymerases have a proofreading activity. Here, the polymerase recognizes the occasional mistakes in the synthesis reaction by the lack of base pairing between the mismatched nucleotides. If a mismatch is detected, a 3′ to 5′ exonuclease activity is activated and the incorrect base removed.^[82] In most organisms DNA polymerases function in a large complex called the replisome that contains multiple accessory subunits, such as the DNA clamp or helicases.^[83]

RNA-dependent DNA polymerases are a specialized class of polymerases that copy the sequence of an RNA strand into DNA. They include reverse transcriptase, which is a viral enzyme involved in the infection of cells by retroviruses, and telomerase, which is required for the replication of telomeres.^[84][32] Telomerase is an unusual polymerase because it contains its own RNA template as part of its structure.^[33]

Transcription is carried out by a DNA-dependent RNA polymerase that copies the sequence of a DNA strand into RNA. To begin transcribing a gene, the RNA polymerase binds to a sequence of DNA called a promoter and separates the DNA strands. It then copies the gene sequence into a messenger RNA transcript until it reaches a region of DNA called the terminator, where it halts and detaches from the DNA. As with human DNA-dependent DNA polymerases, RNA polymerase II, the enzyme that transcribes most of the genes in the human genome, operates as part of a large protein complex with multiple regulatory and accessory subunits.^[85]

http://en.wikipedia.org/wiki/DNA

Wiring Up DNA

Measuring the conductivity of DNA could provide a way to detect mutations.

Hot-wired: By placing a double-stranded DNA segment in a gap in a single-walled carbon nanotube, researchers have measured the electrical properties of the biological molecule. Since even a single mismatch in the DNA letters affects the conductivity of the segment, the system could eventually be the basis of chemical sensors to detect mutations in DNA. Credit: Colin Nuckolls

By wiring up DNA between two carbon nanotubes, researchers have measured the molecule's ability to conduct electricity. Introducing just a single letter change can drastically alter the DNA's resistance, the researchers found, a phenomenon that they plan to exploit with a device that can rapidly screen DNA for disease-linked mutations.

Measuring the electrical properties of DNA has proved tricky because the molecule and its attachments to electrodes tend to be very fragile. But in the new study, Colin Nuckolls, a professor of chemistry at Columbia University, in New York, teamed up with Jacqueline Barton, a professor of chemistry at Caltech, in Pasadena, CA, who's an expert in DNA charge transport. Nuckolls's group had previously developed a method for securely hooking up biological molecules to single-walled carbon nanotubes, which act as the electrodes in a miniscule circuit.

The researchers used an etching process to slice a gap in a carbon nanotube; they created a carboxylic acid group on the nanotube at each end of the gap. They then reacted these groups with DNA strands whose ends had been tagged with amine groups, creating tough chemical amide links that bond together the nanotubes and DNA. The amide linkages are robust enough to withstand enormous electrical fields.

The team estimated that DNA strands of around 15 base pairs (around 6 nanometers) in length had a resistance roughly equivalent to that of a similar-sized piece of graphite. This is a finding that the researchers might have expected since the chemical base pairs that constitute DNA create a stack of aromatic rings similar to those in graphite.

"In my opinion, the results of this work will survive, in contrast to many other publications on this topic," says chemist Bernd Giese, of the University of Basel, Switzerland. Previous estimates of DNA's conductivity have varied dramatically, Giese says, partly because it was unclear if the delicate DNA or its connection to electrodes had become damaged by the high voltages used. "One thinks one has burned the DNA to charcoal," Giese says. "It's extremely complicated experimentally."

Barton and Nuckolls performed two tricks with their wired-up DNA. For their first, they introduced a restriction enzyme that bound and cut the DNA at a specific sequence. When severed, the current running through the DNA vanished. "It's a way of biochemically blowing a fuse," Nuckolls says. It also demonstratesthat the DNA keeps its native structure in the circuit; if it had not, the enzyme would not recognize and cut the molecule.

For their second trick, the researchers introduced a single base-pair mismatch into the DNA so that, for example, a C was paired up with an A (rather than its normal partner, G). This tweak boosted the molecule's resistance some 300-fold, probably because it distorts the double helical structure. They could do this easily by connecting only one of DNA's two strands into the circuit. The second strand - which can either be a perfect match to the first or contain a mismatch - can lift on or off.

Showing the electrical effect of such sequence mismatch and enzyme cutting is the real strength of the experiments, says Danny Porath, of Hebrew University, in Jerusalem, Israel, who has also measured current through DNA. "They play with the parameters and show that conductivity of DNA clearly depends on them, and that's beautiful," he says.

Nuckolls is now working to exploit this discovery to detect single nucleotide polymorphisms (SNPs), the one-letter variations in DNA that are linked to, for example, susceptibility to Alzheimer's, diabetes, and many other major diseases. Nuckolls hopes that his method can be used to identify SNPs more rapidly and with greater sensitivity than existing methods. In such a device, a reference strand of DNA is wired into the circuit and other strands allowed to pair up with it. If the second strand carries a different base at the position of the SNP, this would be enough to trigger a change in the current through a nanoscale circuit, just as the base-pair mismatch did. Nuckolls says that he is already working with electrical engineers to create a sensor that can slot into existing semiconductor chips, making it cheap and readily available. "It's one of our big focuses, and we're pretty close," he says.

The team is likely to have competition. Late last year, a group led by Wonbong Choi, of Florida International University, in Miami, reported that it had strung 80 base pairs of DNA between two carbon nanotubes and sent current through the DNA. Choi says that he is working to create a sensor that can rapidly reveal the presence of specific genetic sequences--such as the avian influenza virus--by looking at changes in current through the tiny circuit.

Barton, meanwhile, is intent on finding out whether the conductivity of DNA serves any biological purpose in the cell. She has evidence that proteins bound to DNA may detect DNA damage by changes in its electrical properties, perhaps triggering repair of the damage. "We think it's something nature takes advantage of," she says. "It's a radical idea, but I think as we get more and more evidence, the case will be built."

http://www.technologyreview.com/Nanotech/20205/

Genes and genomes

Further information: Cell nucleus, Chromatin, Chromosome, Gene, Noncoding DNA

Genomic DNA is located in the cell nucleus of eukaryotes, as well as small amounts in mitochondria and chloroplasts. In prokaryotes, the DNA is held within an irregularly shaped body in the cytoplasm called the nucleoid.^[54] The genetic information in a genome is held within genes, and the complete set of this information in an organism is called its genotype. A gene is a unit of heredity and is a region of DNA that influences a particular characteristic in an organism. Genes contain an open reading frame that can be transcribed, as well as regulatory sequences such as promoters and enhancers, which control the transcription of the open reading frame.

In many species, only a small fraction of the total sequence of the genome encodes protein. For example, only about 1.5% of the human genome consists of protein-coding exons, with over 50% of human DNA consisting of non-coding repetitive sequences.^[55] The reasons for the presence of so much non-coding DNA in eukaryotic genomes and the extraordinary differences in genome size, or C-value, among species represent a long-standing puzzle known as the "C-value enigma."^[56] However, DNA sequences that do not code protein may still encode functional non-coding RNA molecules, which are involved in the regulation of gene expression.^[57]

T7 RNA polymerase (blue) producing a mRNA (green) from a DNA template (orange).^[58]

Some non-coding DNA sequences play structural roles in chromosomes. Telomeres and centromeres typically contain few genes, but are important for the function and stability of chromosomes.^[33][59] An abundant form of non-coding DNA in humans are pseudogenes, which are copies of genes that have been disabled by mutation.^[60] These sequences are usually just molecular fossils, although they can occasionally serve as raw genetic material for the creation of new genes through the process of gene duplication and divergence.^[61]

Transcription and translation

Further information: Genetic code, Transcription (genetics), Protein biosynthesis

A gene is a sequence of DNA that contains genetic information and can influence the phenotype of an organism. Within a gene, the sequence of bases along a DNA strand defines a messenger RNA sequence, which then defines one or more protein sequences. The relationship between the nucleotide sequences of genes and the amino-acid sequences of proteins is determined by the rules of translation, known collectively as the genetic code. The genetic code consists of three-letter 'words' called codons formed from a sequence of three nucleotides (e.g. ACT, CAG, TTT).

In transcription, the codons of a gene are copied into messenger RNA by RNA polymerase. This RNA copy is then decoded by a ribosome that reads the RNA sequence by base-pairing the messenger RNA to transfer RNA, which carries amino acids. Since there are 4 bases in 3-letter combinations, there are 64 possible codons (4³ combinations). These encode the twenty standard amino acids, giving most amino acids more than one possible codon. There are also three 'stop' or 'nonsense' codons signifying the end of the coding region; these are the TAA, TGA and TAG codons.

DNA replication. The double helix is unwound by a helicase and topoisomerase. Next, one DNA polymerase produces the leading strand copy. Another DNA polymerase binds to the lagging strand. This enzyme makes discontinuous segments (called Okazaki fragments) before DNA ligase joins them together.

Replication

Further information: DNA replication

Cell division is essential for an organism to grow, but when a cell divides it must replicate the DNA in its genome so that the two daughter cells have the same genetic information as their parent. The double-stranded structure of DNA provides a simple mechanism for DNA replication. Here, the two strands are separated and then each strand's complementary DNA sequence is recreated by an enzyme called DNA polymerase. This enzyme makes the complementary strand by finding the correct base through complementary base pairing, and bonding it onto the original strand. As DNA polymerases can only extend a DNA strand in a 5′ to 3′ direction, different mechanisms are used to copy the antiparallel strands of the double helix.^[62] In this way, the base on the old strand dictates which base appears on the new strand, and the cell ends up with a perfect copy of its DNA.

Interactions with proteins

All the functions of DNA depend on interactions with proteins. These protein interactions can be non-specific, or the protein can bind specifically to a single DNA sequence. Enzymes can also bind to DNA and of these, the polymerases that copy the DNA base sequence in transcription and DNA replication are particularly important.

http://en.wikipedia.org/wiki/DNA

Molecular and biomolecular monolayers on diamond as an interface to biology

Covalent modification of diamond surfaces with molecular monolayers serves as a starting point for linking biomolecules such as DNA and proteins to surfaces. DNA-modified diamond surfaces show excellent selectivity for hybridization with complementary vs. noncomplementary sequences and outstanding stability. These biomolecular recognition events also lead to changes in the conductivity of the interface via a molecular field effect in which the negative charge on the DNA molecules alters the conductivity in the diamond thin film. Chemical modification of the diamond with ethylene glycol oligomers leads to surfaces that effectively resist the nonspecific adsorption of proteins and other biomolecules to the surface. These outstanding physical and chemical properties of chemically modified diamond surfaces present new opportunities for the use of diamond thin films in biological applications.

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TWV-4DTTMB0-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=60543ee8ac4dfe091837fe2d3cc66231

DNA Chemical modifications

Chemical modifications

cytosine	5-methylcytosine	thymine

Structure of cytosine with and without the 5-methyl group. After deamination the 5-methylcytosine has the same structure as thymine

Base modifications

Further information: DNA methylation

The expression of genes is influenced by the chromatin structure of a chromosome and regions of that have low or no gene expression usually contain high levels of methylation of cytosine bases. For example, cytosine methylation, producing 5-methylcytosine, is important for X-chromosome inactivation.^[38] The average level of methylation varies between organisms, with Caenorhabditis elegans lacking cytosine methylation, while vertebrates show higher levels, with up to 1% of their DNA containing 5-methylcytosine.^[39] Despite the biological role of 5-methylcytosine it can deaminate to leave a thymine base, methylated cytosines are therefore particularly prone to mutations.^[40] Other base modifications include adenine methylation in bacteria and the glycosylation of uracil to produce the "J-base" in kinetoplastids.^[41][42]

DNA damage

Further information: Mutation

Benzopyrene, the major mutagen in tobacco smoke, in an adduct to DNA^[43]

DNA can be damaged by many different sorts of mutagens, which are agents that change the DNA sequence. These agents include oxidizing agents, alkylating agents and also high-energy electromagnetic radiation such as ultraviolet light and X-rays. The type of DNA damage produced depends on the type of mutagen. For example, UV light mostly damages DNA by producing thymine dimers, which are cross-links between adjacent pyrimidine bases in a DNA strand.^[44] On the other hand, oxidants such as free radicals or hydrogen peroxide produce multiple forms of damage, including base modifications, particularly of guanosine, as well as double-strand breaks.^[45] It has been estimated that in each human cell, about 500 bases suffer oxidative damage per day.^[46][47] Of these oxidative lesions, the most dangerous are double-strand breaks, as these are difficult to repair and can produce point mutations, insertions and deletions from the DNA sequence, as well as chromosomal translocations.^[48]

Many mutagens intercalate into the space between two adjacent base pairs. Intercalators are mostly aromatic and planar molecules, and include ethidium, daunomycin, doxorubicin and thalidomide. In order for an intercalator to fit between base pairs, the bases must separate, distorting the DNA strands by unwinding of the double helix. These structural changes inhibit both transcription and DNA replication, causing toxicity and mutations. As a result, DNA intercalators are often carcinogens, with benzopyrene diol epoxide, acridines, aflatoxin and ethidium bromide being well-known examples.^[49][50][51] Nevertheless, due to their properties of inhibiting DNA transcription and replication, they are also used in chemotherapy to inhibit rapidly-growing cancer cells.^[52]

Overview of biological functions

DNA usually occurs as linear chromosomes in eukaryotes, and circular chromosomes in prokaryotes. The set of chromosomes in a cell makes up its genome; the human genome has approximately 3 billion base pairs of DNA arranged into 46 chromosomes.^[53] The information carried by DNA is held in the sequence of pieces of DNA called genes. Transmission of genetic information in genes is achieved via complementary base pairing. For example, in transcription, when a cell uses the information in a gene, the DNA sequence is copied into a complementary RNA sequence through the attraction between the DNA and the correct RNA nucleotides. Usually, this RNA copy is then used to make a matching protein sequence in a process called translation which depends on the same interaction between RNA nucleotides. Alternatively, a cell may simply copy its genetic information in a process called DNA replication. The details of these functions are covered in other articles; here we focus on the interactions between DNA and other molecules that mediate the function of the genome.

http://en.wikipedia.org/wiki/DNA