DNA replication is the process of copying a double-stranded DNA molecule to form two double-stranded molecules. Deoxyribonucleic acid ( DNA) is a Nucleic acid that contains the genetic instructions used in the development and functioning of all known [1][2] The process of DNA replication is a fundamental process used by all living organisms as it is the basis for biological inheritance. As each DNA strand holds the same genetic information, both strands can serve as templates for the reproduction of the opposite strand. The template strand is preserved in its entirety and the new strand is assembled from nucleotides. Nucleotides are Organic compounds that consist of three joined structures a nitrogenous base a Sugar, and a Phosphate group This process is called "semiconservative replication". Semiconservative replication describes the method by which DNA is replicated in all known cells The resulting double-stranded DNA molecules are identical; proofreading and error-checking mechanisms exist to ensure near perfect fidelity.
In a cell, DNA replication must happen before cell division can occur. The cell is the structural and functional unit of all known living Organisms It is the smallest unit of an organism that is classified as living and is often called Cell division is a process by which a cell, called the parent cell divides into two or more cells called daughter cells. DNA synthesis begins at specific locations in the genome, called "origins", where the two strands of DNA are separated. The Origin of replication (also called the replication origin) is a particular sequence in a Genome at which replication is initiated [3] RNA primers attach to single stranded DNA and the enzyme DNA polymerase extends the primers to form new strands of DNA, adding nucleotides matched to the template strand. Enzymes are Biomolecules that catalyze ( ie increase the rates of Chemical reactions Almost all enzymes are Proteins A DNA Polymerase is an Enzyme that assists in DNA replication. The unwinding of DNA and synthesis of new strands forms a replication fork. The replication fork is a structure that forms during DNA replication. In addition to DNA polymerase, a number of other proteins are associated with the fork and assist in the initiation and continuation of DNA synthesis. Proteins are large Organic compounds made of Amino acids arranged in a linear chain and joined together by Peptide bonds between the Carboxyl
DNA replication can also be performed artificially, using the same enzymes used within the cell. DNA polymerases and artificial DNA primers are used to initiate DNA synthesis at known sequences in a template molecule. The polymerase chain reaction (PCR), a common laboratory technique, employs artificial synthesis in a cyclic manner to rapidly and specifically amplify a target DNA fragment from a pool of DNA.
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DNA structure
DNA usually exists in a double-stranded structure, with both strands coiled together to form the characteristic double-helix. In Geometry a double helix (plural helices) typically consists of two congruent helices with the same axis differing by a translation Each single strand of DNA is a chain of four types of nucleotide: adenine, cytosine, guanine, and thymine. Nucleotides are Organic compounds that consist of three joined structures a nitrogenous base a Sugar, and a Phosphate group A nucleotide consists of a phosphate and a deoxyribose sugar forming the backbone of the DNA double helix plus a base that points inwards. Nucleotides are matched between strands through hydrogen bonds to form base pairs. In Molecular biology, two Nucleotides on opposite complementary DNA or RNA strands that are connected via Hydrogen bonds are called Adenine pairs with thymine and cytosine pairs with guanine. Adenine is a Purine with a variety of roles in Biochemistry including Cellular respiration, in the form of both the energy-rich Adenosine Thymine is one of the four bases in the Nucleic acid of DNA that make up the letters ATGC Cytosine is one of the five main bases found in DNA and RNA. It is a Pyrimidine derivative with a Heterocyclic Aromatic ring Guanine is one of the five main Nucleobases found in the Nucleic acids DNA and RNA, the others being Adenine, Cytosine,
The physical pairing of bases in DNA means that the information contained within each strand is redundant. The nucleotides on a single strand can be used to reconstruct nucleotides on a newly synthesized partner strand.
DNA strands have a directionality, and the different ends of a single strand are called the "3' end" and the "5' end" (these refer to the carbon atom in ribose that the next phosphate in the chain attaches to). In addition to being complementary, the two strands of DNA are antiparallel: they are orientated in opposite directions. This directionality has consequences in DNA synthesis, because DNA polymerase can only synthesize DNA in one direction by adding nucleotides to the 3' end of a DNA strand.
DNA polymerase
DNA polymerases are a family of enzymes critical for all forms of DNA replication. A DNA Polymerase is an Enzyme that assists in DNA replication. Enzymes are Biomolecules that catalyze ( ie increase the rates of Chemical reactions Almost all enzymes are Proteins [4] A DNA polymerase synthesizes a new strand of DNA by extending the 3' end of an existing nucleotide chain, adding new nucleotides matched to the template strand one at a time. Nucleotides are Organic compounds that consist of three joined structures a nitrogenous base a Sugar, and a Phosphate group Some DNA polymerases may also have some proofreading ability, removing nucleotides from the end of a strand in order to remove any mismatched bases. DNA polymerases are generally extremely accurate, making less than one error for every million nucleotides added.
The energy for the process of DNA polymerization comes from the two additional phosphates attached to each of the unincorporated nucleotides. These free nucleotides, also known as nucleoside triphosphates, contain a total of three phosphates. Nucleoside triphosphate (NTP is a Nucleoside with three Phosphates Natural nucleoside triphosphates include Adenosine triphosphate (ATP Guanosine When a nucleotide is being added to a growing DNA strand, two of the phosphates are removed and the energy produced is used to attach the remaining phosphate to the growing chain. The energetics of this process may also explain the directionality of synthesis - if DNA were synthesized in the 3' to 5' direction, the energy for the process would come from the 5' end of the growing strand rather than from free nucleotides. During proofreading, if the 5' nucleotide needed to be removed this triphosphate end would be lost, losing the energy source required to add a new nucleotide to the end.
DNA polymerase can only extend an existing DNA strand paired with a template strand, it cannot begin the synthesis of a new strand. To do this a short fragment of DNA or RNA, called a primer, must be created and paired with the template strand before DNA polymerase can synthesize new DNA. Ribonucleic acid ( RNA) is a Nucleic acid that consists of a long chain of Nucleotide units
DNA replication within the cell
Origins of replication
For a cell to divide, it must first replicate its DNA. DNA replication in Prokaryotes is exemplified in E coli. It is bi-directional and originates at a single Origin of replication Although the mechanisms of DNA synthesis in Eukaryotes and Prokaryotes are similar DNA replication in eukaryotes is much more complicated [5] This process is initiated at particular points within the DNA, known as "origins", which are targeted by proteins that separate the two strands and initiate DNA synthesis. The Origin of replication (also called the replication origin) is a particular sequence in a Genome at which replication is initiated [3] Origins contain DNA sequences recognized by replication initiator proteins (eg. dnaA in E coli' and the Origin Recognition Complex in yeast). DnaA is a Replication Initiation factor which promotes the unwinding or denaturation of DNA at OriC (around 240 bp in ORC or Origin Recognition Complex is a multisubunit complex (6 subunits existing in the replication procedure of DNA. [6] These initiator proteins recruit other proteins to separate the two strands and initiate replication forks.
Initiator proteins recruit other proteins to separate the DNA strands at the origin, forming a bubble. Origins tend to be "AT-rich" (rich in adenine and thymine bases) to assist this process because A-T base pairs have two hydrogen bonds (rather than the three formed in a C-G pair)—strands rich in these nucleotides are generally easier to separate. [7] Once strands are separated, RNA primers are created on the template strands and DNA polymerase extends these to create newly synthesized DNA.
As DNA synthesis continues, the original DNA strands continue to unwind on each side of the bubble, forming replication forks. The replication fork is a structure that forms during DNA replication. In bacteria, which have a single origin of replication on their circular chromosome, this process eventually creates a "theta structure" (resembling the Greek letter theta: θ). A Theta structure is an intermediate structure formed during the replication of a circular DNA Molecule (prokaryote DNA two replication forks can proceed In contrast, eukaryotes have longer linear chromosomes and initiate replication at multiple origins within these.
The replication fork
The replication fork is a structure which forms when DNA is being replicated. It is created through the action of helicase, which breaks the hydrogen bonds holding the two DNA strands together. Helicases are a class of Enzymes vital to all living Organisms They are motor proteins that move directionally along a Nucleic acid phosphodiester backbone The resulting structure has two branching "prongs", each one made up of a single strand of DNA.
- Leading strand synthesis
In DNA replication, the leading strand is defined as the new DNA strand at the replication fork that is synthesized in the 5'→3' direction in a continuous manner. The replication fork is a structure that forms during DNA replication. When the enzyme helicase unwinds DNA, two single stranded regions of DNA (the "replication fork") form. On the leading strand DNA polymerase III is able to synthesize DNA using the free 3' OH group donated by a single RNA primer and continuous synthesis occurs in the direction in which the replication fork is moving. Pol III can also refer to KNM Pol III, a Norwegian guard vessel from WW2 DNA polymerase III holoenzyme is the primary Enzyme complex
- Lagging strand synthesis
The lagging strand is the DNA strand at the opposite side of the replication fork from the leading strand, running in the 3' to 5' direction. The replication fork is a structure that forms during DNA replication. Because DNA polymerase cannot synthesize in the 3'→5' direction, the lagging strand is synthesized in short segments known as Okazaki fragments. An Okazaki fragment is a relatively short fragment of DNA (with an RNA primer at the 5' terminus created on the Lagging strand during DNA replication. Along the lagging strand's template, primase builds RNA primers in short bursts. DNA primase is an Enzyme involved in the replication of DNA by creating an RNA primer DNA polymerases are then able to use the free 3' OH groups on the RNA primers to synthesize DNA in the 5'→3' direction. The RNA fragments are then removed (different mechanisms are used in eukaryotes and prokaryotes) and new deoxyribonucleotides are added to fill the gaps where the RNA was present. DNA ligase then joins the deoxyribonucleotides together, completing the synthesis of the lagging strand. In Molecular biology, DNA ligase is a special type of Ligase ( that can link together two DNA strands that have single-strand breaks (a break in both complementary
- Dynamics at the replication fork
As helicase unwinds DNA at the replication fork, the DNA ahead is forced to rotate. This process results in a build-up of twists in the DNA ahead. [8] This build-up would form a resistance that would eventually halt the progress of the replication fork. DNA topoisomerases are enzymes that solve these physical problems in the coiling of DNA. Topoisomerases (type I, type II) are Isomerase Enzymes that act on the topology of DNA. Topoisomerase I cuts a single backbone on the DNA, enabling the strands to swivel around each other to remove the build-up of twists. Topoisomerase II cuts both backbones, enabling one double-stranded DNA to pass through another, thereby removing knots and entanglements that can form within and between DNA molecules.
Bare single-stranded DNA has a tendency to fold back upon itself and form secondary structures; these structures can interfere with the movement of DNA polymerase. In Biochemistry and Structural biology, secondary structure is the general three-dimensional form of local segments of Biopolymers such as To prevent this, single-strand binding proteins bind to the DNA until a second strand is synthesized, preventing secondary structure formation. Single-strand binding protein, also known as SSB or SSBP binds single stranded regions of DNA to prevent premature reannealing [9]
Clamp proteins form a sliding clamp around DNA, helping the DNA polymerase maintain contact with its template and thereby assisting with processivity. A DNA clamp, also known as a sliding clamp, is a Protein fold that serves as a Processivity -promoting factor in DNA replication. The inner face of the clamp enables DNA to be threaded through it. Once the polymerase reaches the end of the template or detects double stranded DNA, the sliding clamp undergoes a conformational change which releases the DNA polymerase. Clamp-loading proteins are used to initially load the clamp, recognizing the junction between template and RNA primers.
Regulation of replication
- Eukaryotes
Within eukaryotes, DNA replication is controlled within the context of the cell cycle. The cell cycle, or cell-division cycle, is the series of events that take place in a eukaryotic cell leading to its replication As the cell grows and divides, it progresses through stages in the cell cycle; DNA replication occurs during the S phase (Synthesis phase). The progress of the eukaryotic cell through the cycle is controlled by cell cycle checkpoints. Cell cycle checkpoints are control mechanisms that ensure the fidelity of cell division in Eukaryotic cells. Progression through checkpoints is controlled through complex interactions between various proteins, including cyclins and cyclin-dependent kinases. Cyclins are a family of Proteins involved in the progression of cells through the Cell cycle. Cyclin-dependent kinases ( CDK) belong to a group of Protein kinases originally discovered as being involved in the regulation of the Cell cycle. [10]
The G1/S checkpoint (or restriction checkpoint) regulates whether eukaryotic cells enter the process of DNA replication and subsequent division. Cells which do not proceed through this checkpoint are quiescent in the "G0" stage and do not replicate their DNA.
Replication of chloroplast and mitochondrial genomes occurs independent of the cell cycle, through the process of D-loop replication. D-loop replication is a process by which Chloroplasts and Mitochondria replicate their genetic material
- Bacteria
Most bacteria do not go through a well-defined cell cycle and instead continuously copy their DNA; during rapid growth this can result in multiple rounds of replication occurring concurrently. [11] Within E coli, the most well-characterized bacteria, regulation of DNA replication can be achieved through several mechanisms, including: the hemimethylation and sequestering of the origin sequence, the ratio of ATP to ADP, and the levels of protein DnaA. These all control the process of initiator proteins binding to the origin sequences.
Because E coli methylates GATC DNA sequences, DNA synthesis results in hemimethylated sequences. DNA methylation is a type of chemical modification of DNA that can be inherited and subsequently removed without changing the original DNA sequence This hemimethylated DNA is recognized by a protein (SeqA) which binds and sequesters the origin sequence; in addition, dnaA (required for initiation of replication) binds less well to hemimethylated DNA. As a result, newly replicated origins are prevented from immediately initiating another round of DNA replication. [12]
ATP builds up when the cell is in a rich medium, triggering DNA replication once the cell has reached a specific size. ATP competes with ADP to bind to DnaA, and the DNA-ATP complex is able to initiate replication. A certain number of DnaA proteins are also required for DNA replication — each time the origin is copied the number of binding sites for DnaA doubles, requiring the synthesis of more DnaA to enable another initiation of replication.
Termination of replication
Because bacteria have circular chromosomes, termination of replication occurs when the two replication forks meet each other on the opposite end of the parental chromosome. E coli regulate this process through the use of termination sequences which, when bound by the Tus protein, enable only one direction of replication fork to pass through. As a result, the replication forks are constrained to always meet within the termination region of the chromosome. [13]
Eukaryotes initiate DNA replication at multiple points in the chromosome, so replication forks meet and terminate at many points in the chromosome; these are not known to be regulated in any particular manner. Because eukaryotes have linear chromosomes, DNA replication often fails to synthesize to the very end of the chromosomes (telomeres), resulting in telomere shortening. A telomere is a region of repetitive DNA at the end of Chromosomes which protects the end of the chromosome from destruction This is a normal process in somatic cells — cells are only able to divide a certain number of times before the DNA loss prevents further division. Somatic cells are any cells forming the body of an organism as opposed to Germline cells (This is known as the Hayflick limit. The Hayflick limit is the number of times a cell will divide before it stops due to the telomere reaching a critical length. ) Within the germ cell line, which passes DNA to the next generation, the enzyme telomerase extends the repetitive sequences of the telomere region to prevent degradation. Germ cells are progenitors of the Gametes. These singled out cells move through the gut to the developing Gonads and undergo mitotic proliferation followed Telomerase is an Enzyme that adds specific DNA sequence repeats ("TTAGGG" in all vertebrates to the 3' ("three prime" end of DNA strands in the Telomerase can become mistakenly active in somatic cells, sometimes leading to cancer formation. Cancer (medical term Malignant Neoplasm) is a class of Diseases in which a group of cells display uncontrolled
Rolling circle replication
Another method of copying DNA, sometimes used in vivo by bacteria and viruses, is the process of rolling circle replication. Rolling circle replication describes a process of nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as In vivo ( Latin: within the living means that which takes place inside an organism. Rolling circle replication describes a process of nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as [14] In this form of replication, a single replication fork progresses around a circular molecule to form multiple linear copies of the DNA sequence. In cells, this process can be used to rapidly synthesize multiple copies of plasmids or viral genomes.
In the cell, rolling circle replication is initiated by an initiator protein encoded by the plasmid or virus DNA. This protein is able to nick one strand of the double-stranded, circular DNA molecule at a site called the double-strand origin (DSO) and remains bound to the 5' phosphate end of the nicked strand. The free 3' hydroxyl end is released and can serve as a primer for DNA synthesis. Using the unnicked strand as a template, replication proceeds around the circular DNA molecule, displacing the nicked strand as single-stranded DNA. Continued DNA synthesis produces multiple single-stranded linear copies of the original DNA in a continuous head-to-tail series. In vivo these linear copies are subsequently converted to double-stranded circular molecules.
Rolling circle replication can also be performed in vitro and has found wide uses in academic research and biotechnology, often used for amplification of DNA from very small amounts of starting material. In vitro ( Latin: within the glass refers to the technique of performing a given experiment in a controlled environment outside of a living Organism Replication can be initiated by nicking a double-stranded circular DNA molecule or by hybridizing a primer to a single-stranded circle of DNA. The use of a reverse primer (or random primers) produces hyperbranched rolling circle amplification, resulting in exponential rather than linear growth of the DNA molecule.
Polymerase chain reaction
In vitro, researchers commonly replicate DNA using the polymerase chain reaction (PCR). PCR uses a pair of primers to span a target region in template DNA, polymerizing partner strands in each direction. This process can be repeated through multiple cycles through the use of a thermostable polymerase. At the start of each cycle, the mixture of template and primers is heated, separating the newly synthesized molecule and template. Then, as the mixture cools, both of these become templates for new primers to anneal to, and the polymerase extends from these. As a result the number of copies of the target region doubles each round, growing exponentially.
See also
References
- ^ Berg JM, Tymoczko JL, Stryer L, Clarke ND (2002). A replicon is a DNA molecule or RNA molecule or a region of DNA or RNA that replicates from a single Origin of replication. Replicative transposition is a mechanism of transposition in Molecular biology in which the Transposable element is duplicated during the reaction so that the transposing The replication of the DNA of Escherichia coli proceeds via the replisome, a multiprotein workhorse that in other organisms may vary in complexity See also Biological reproduction Self-replication is any process by which a thing might make a copy of itself Chronic since 1992 ( talk) 2200 12 December 2007 (UTC --> Viral replication Biochemistry. W. H. Freeman and Company. ISBN 0-7167-3051-0. Chapter 27: DNA Replication, Recombination, and Repair
- ^ Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002). Molecular Biology of the Cell. Garland Science. ISBN 0-8153-3218-1. Chapter 5: DNA Replication, Repair, and Recombination
- ^ a b Berg JM, Tymoczko JL, Stryer L, Clarke ND (2002). Biochemistry. W. H. Freeman and Company. ISBN 0-7167-3051-0. Chapter 27, Section 4: DNA Replication of Both Strands Proceeds Rapidly from Specific Start Sites
- ^ Berg JM, Tymoczko JL, Stryer L, Clarke ND (2002). Biochemistry. W. H. Freeman and Company. ISBN 0-7167-3051-0. Chapter 27, Section 2: DNA Polymerases Require a Template and a Primer
- ^ Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002). Molecular Biology of the Cell. Garland Science. ISBN 0-8153-3218-1. Chapter 5: DNA Replication Mechanisms
- ^ C Weigel, A Schmidt, B Rückert, R Lurz, and W Messer (1997). "DnaA protein binding to individual DnaA boxes in the Escherichia coli replication origin, oriC". EMBO Journal 16 (21): 6574–6583. doi:. A digital object identifier ( DOI) is a permanent identifier given to an Electronic document. PMID 9351837.
- ^ Lodish H, Berk A, Zipursky LS, Matsudaira P, Baltimore D, Darnell J (2000). Molecular Cell Biology. W. H. Freeman and Company. ISBN 0-7167-3136-3. 12.1. General Features of Chromosomal Replication: Three Common Features of Replication Origins
- ^ Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002). Molecular Biology of the Cell. Garland Science. ISBN 0-8153-3218-1. DNA Replication Mechanisms: DNA Topoisomerases Prevent DNA Tangling During Replication
- ^ Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002). Molecular Biology of the Cell. Garland Science. ISBN 0-8153-3218-1. DNA Replication Mechanisms: Special Proteins Help to Open Up the DNA Double Helix in Front of the Replication Fork
- ^ Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002). Molecular Biology of the Cell. Garland Science. ISBN 0-8153-3218-1. Intracellular Control of Cell-Cycle Events: S-Phase Cyclin-Cdk Complexes (S-Cdks) Initiate DNA Replication Once Per Cycle
- ^ Tobiason DM, Seifert HS (2006). "The Obligate Human Pathogen, Neisseria gonorrhoeae, Is Polyploid". PLoS Biology 4 (6).
- ^ Slater S, Wold S, Lu M, Boye E, Skarstad K, Kleckner N (1995). "E. coli SeqA protein binds oriC in two different methyl-modulated reactions appropriate to its roles in DNA replication initiation and origin sequestration.". Cell 82 (6): 927-36. doi:. A digital object identifier ( DOI) is a permanent identifier given to an Electronic document. PMID 7553853.
- ^ TA Brown (2002). Genomes. BIOS Scientific Publishers Ltd. ISBN 1 85996 228 9. 13.2.3. Termination of replication
- ^ Griffiths AJF, Miller JH, Suzuki DT, Lewontin RC, Gelbart WM (2000). An Introduction to Genetic Analysis. W. H. Freeman. ISBN 0-7167-3520-2. Chapter 8 Replication of DNA: Rolling-circle replication
External links
- DNA Replication Flash Animation DNA Replication tutorial animation
- DNA Workshop
- WEHI-TV - DNA Replication video Detailed DNA replication animation from different angles with description below.
- Breakfast of Champions Does Replication Creative primer on the process from the Science Creative Quarterly
- Basic Polymerase Chain Reaction Protocol
- Animated Biology
- DNA makes DNA (Flash Animation)
- DNA replication info page by George Kakaris, Biologist MSc in Applied Genetics and Biotechnology
- Reference website on eukaryotic DNA replication
- Molecular visualization of DNA Replication
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