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وفاء عبد الواحد.د المحاضرة االولى والثانية Microbial genetics is a subject area within microbiology and genetic engineering. It studies the genetics of very small (micro) organisms; bacteria, archaea, viruses and some protozoa and fungi. This involves the study of the genotype of microbial species and also the expression system in the form of phenotypes. Since the discovery of microorganisms by two Fellows of The Royal Society, Robert Hooke and Antoni van Leeuwenhoek during the period 1665-1885they have been used to study many processes and have had applications in various areas of study in genetics. For example: Microorganisms' rapid growth rates and short generation times are used by scientists to study evolution.[] Microbial genetics also has applications in being able to study processes and pathways that are similar to those found in humans such as drug metabolism.] Bacter Bacteria are classified by their shape. Bacteria have been on this planet for approximately 3.5 billion years, and are classified by their shape.[5] Bacterial genetics studies the mechanisms of their heritable information, their chromosomes, plasmids, transposons, and phages.[6] وفاء عبد الواحد.د المحاضرة االولى والثانية Gene transfer systems that have been extensively studied in bacteria include genetic transformation, conjugation and transduction.Natural transformation is a bacterial adaptation for DNA transfer between two cells through the intervening medium. The uptake of donor DNA and its recombinational incorporation into the recipient chromosome depends on the expression of numerous bacterial genes whose products direct this process. In general, transformation is a complex, energy-requiring developmental process that appears to be an adaptation for repairing DNA damage. Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cellto-cell contact or by a bridge-like connection between two cells. Bacterial conjugation has been extensively studied in Escherichia coli, but also occurs in other bacteria such asMycobacterium smegmatis. Conjugation requires stable and extended contact between a donor and a recipient strain, is DNase resistant, and the transferred DNA is incorporated into the recipient chromosome by homologous recombination. E. coli conjugation is mediated by expression of plasmid genes, whereas mycobacterial conjugation is mediated by genes on the bacterial chromosome Transduction is the process by which foreign DNA is introduced into a cell by a virus or viral vector. Transduction is a common tool used by molecular biologists to stably introduce a foreign gene into a host cell's genome Archaea[edit] Archaea is a domain of organisms that are prokaryotic, single-celled, and are thought to have developed 4 billion years ago. They share a common وفاء عبد الواحد.د المحاضرة االولى والثانية ancestor with bacteria, but are more closely related to eukaryotes in comparison to bacteria.] Some Archaea are able to survive extreme environments, which leads to many applications in the field of genetics. One of such applications is the use of archaeal enzymes, which would be better able to survive harsh conditions in vitro. Gene transfer and genetic exchange have been studied in the halophilic archaeon Halobacterium volcanii and the hyperthermophilic archaeons Sulfolobus solfataricus andSulfolobus acidocaldarius. H. volcani forms cytoplasmic bridges between cells that appear to be used for transfer of DNA from one cell to another in either direction.] When S. solfataricus and S. acidocaldarius are exposed to DNA damaging agents, species-specific cellular aggregation is induced. Cellular aggregation mediates chromosomal marker exchange and genetic recombination with high frequency. Cellular aggregation is thought to enhance species specific DNA transfer between Sulfolobus cells in order to provide increased repair of damaged DNA by means of homologous recombination.[14][15][16] Fungi[edit] Fungi can be both multicellular and unicellular organisms, and are distinguished from other microbes by the way they obtain nutrients. Fungi secrete enzymes into their surroundings, to break down organic matter. Fungal genetics uses yeast, and filamentous fungi as model organisms for eukaryotic genetic research, including cell cycleregulation, chromatin structure and gene regulation.[17] Studies of the fungus Neurospora crassa have contributed substantially to understanding how genes work. N. crassa is a type of red bread mold of the phylum Ascomycota. It is used as a model organism because it is easy to grow and has a haploid life cycle that makes genetic analysis simple وفاء عبد الواحد.د المحاضرة االولى والثانية since recessive traits will show up in the offspring. Analysis of genetic recombination is facilitated by the ordered arrangement of the products of meiosis in ascospores. In its natural environment, N. crassa lives mainly in tropical and sub-tropical regions. It often can be found growing on dead plant matter after fires. Neurospora was used by Edward Tatum and George Beadle in their experiments for which they won the Nobel Prize in Physiology or Medicine in 1958. The results of these experiments led directly to the one gene-one enzyme hypothesis that specific genes code for specific proteins. This concept proved to be the opening gun in what becamemolecular genetics and all the developments that have followed from that.[19] Saccharomyces cerevisiae is a yeast of the phylum Ascomycota. During vegetative growth that ordinarily occurs when nutrients are abundant, S. cerevisiae reproduces by mitosisas diploid cells. However, when starved, these cells undergo meiosis to form haploid spores.[20] Mating occurs when haploid cells of opposite mating types MATa and MATα come into contact. Ruderfer et al. pointed out that, in nature, such contacts are frequent between closely related yeast cells for two reasons. The first is that cells of opposite mating type are present together in the same acus, the sac that contains the cells directly produced by a single meiosis, and these cells can mate with each other. The second reason is that haploid cells of one mating type, upon cell division, often produce cells of the opposite mating type. An analysis of the ancestry of natural S. cerevisiae strains concluded that outcrossing occurs very infrequently (only about once every 50,000 cell divisions).[21] The relative rarity in nature of meiotic events that result from outcrossing suggests that the possible long-term benefits of outcrossing (e.g. generation of diversity) وفاء عبد الواحد.د المحاضرة االولى والثانية are unlikely to be sufficient for generally maintaining sex from one generation to the next. Rather, a short term benefit, such as meiotic recombinational repair of DNA damages caused by stressful conditions (such as starvation) may be the key to the maintenance of sex in S. cerevisiae. Candida albicans is a diploid fungus that grows both as a yeast and as a filament. C. albicans is the most common fungal pathogen in humans. It causes both debilitating mucosal infections and potentially lifethreatening systemic infections. C. albicans has maintained an elaborate, but largely hidden, mating apparatus. Johnson suggested that mating strategies may allow C. albicans to survive in the hostile environment of a mammalian host. Among the 250 known species of aspergilli, about 33% have an identified sexual state.] Among those Aspergillus species that exhibit a sexual cycle the overwhelming majority in nature are homothallic (selffertilizing).] Selfing in the homothallic fungus Aspergillus nidulans involves activation of the same mating pathways characteristic of sex in outcrossing species, i.e. self-fertilization does not bypass required pathways for outcrossing sex but instead requires activation of these pathways within a single individual.]Fusion of haploid nuclei occurs within reproductive structures termed cleistothecia, in which the diploid zygote undergoes meiotic divisions to yield haploid ascospores. Viruses Viruses are capsid-encoding organisms composed of proteins and nucleic acids that can self-assemble after replication in a host cell using the host's replication machineryThere is a disagreement in science about whether viruses are living due to their lack وفاء عبد الواحد.د المحاضرة االولى والثانية of ribosomes.] Comprehending the viral genome is important not only for studies in genetics but also for understanding their pathogenic properties.] Many types of virus are capable of genetic recombination. When two or more individual viruses of the same type infect a cell, their genomes may recombine with each other to produce recombinant virus progeny. Both DNA and RNA viruses can undergo recombination. When two or more viruses, each containing lethal genomic damage infect the same host cell, the virus genomes often can pair with each other and undergo homologous recombinational repair to produce viable progeny. This process is known as multiplicity reactivation. Enzymes employed in multiplicity reactivation are functionally homologous to enzymes employed in bacterial and eukaryotic recombinational repair. Multiplicity reactivation has been found to occur with pathogenic viruses including influenza virus, HIV-1, adenovirus simian virus 40, vaccinia virus, reovirus, poliovirus and herpes simplex virus as well as numerous bacteriophages.] Applications of microbial genetics Taq polymerase which is used in Polymerase Chain Reaction(PCR) Microbes are ideally suited for biochemical and genetics studies and have made huge contributions to these fields of science such as the demonstration that DNA is the genetic material,[37][38] that the gene has a وفاء عبد الواحد.د المحاضرة االولى والثانية simple linear structure,] that the genetic code is a triplet code,] and that gene expression is regulated by specific genetic processes.] Jacques Monod and François Jacob used Escherichia coli, a type of bacteria, in order to develop the operon model of gene expression, which lay down the basis of gene expression and regulation.] Furthermore the hereditary processes of single-celled eukaryotic microorganisms are similar to those in multi-cellular organisms allowing researchers to gather information on this process as well.] Another bacterium which has greatly contributed to the field of genetics is Thermus aquaticus, which is a bacterium that tolerates high temperatures. From this microbe scientists isolated the enzyme Taq polymerase, which is now used in the powerful experimental technique, Polymerase chain reaction(PCR).] Additionally the development of recombinant DNA technology through the use of bacteria has led to the birth of modern genetic engineering andbiotechnology.] Using microbes, protocols were developed to insert genes into bacterial plasmids, taking advantage of their fast reproduction, to make biofactories for the gene of interest. Such genetically engineered bacteria can produce pharmaceuticals such as insulin, human growth hormone, interferons and blood clotting factors.[5] Microbes synthesize a variety of enzymes for industrial applications, such as fermented foods, laboratory test reagents, dairy products (such as renin), and even in clothing (such as Trichoderma fungus whose enzyme is used to give jeans a stone washed appearance).[5] Bibliography[edit] وفاء عبد الواحد.د المحاضرة االولى والثانية Genetic recombination contributes to population diversity: recombinations more likely than mutations to provide beneficial change since it tends not to destroy gene function Eukaryotes: recombination during meiosis for sexual reproduction -creates diversity in offspring but parent remains unchanged -vertical gene transfer = genes passed from organism to offspring Prokaryotes: recombination via gene transfer between cells or within cell by transformation, conjugation, or transduction -original cell is altered horizontal gene transfer = genes passed to neighboring microbes of same generation -transfer involves donor cell that gives portion of DNA to recipient cell -when donor DNA incorporated into recipient, recipient now called recombinant cell -if recombinant cell acquired new function/characteristic as result of new DNA, cell has been transformed Generation of recombinant cells is very low frequency event (less than 1%): very few cells in population are capable of exchanging and incorporating DNA Three methods of prokaryotic gene transfer: 1. Bacterial Transformation -genes transferred as naked DNA -can occur between unrelated genus/species -discovered by F. Griffith 1928 who studied Streptococcus pneumoniae -virulent strain had capsule -non-virulent stain did not -in mouse, dead virulent strain could pass “virulence factor” to live nonvirulent strain -competent cells can pick up DNA from dead cells and incorporate it into genome by recombination (e.g. antibiotic resistance) transformed cell than passes genetic recombination to progeny2. Conjugation -genes transferred between two live cells via sex pilus (Gram -) or surface adhesion molecules (Gram +) -transfer mediated by a plasmid: small circle of DNA separate from genome that is self replicating but contains no essential genes -plasmid has genes for its وفاء عبد الواحد.د المحاضرة االولى والثانية own transfer -Gram negative plasmids have genes for pilus -Gram positive plasmids have genes for surface adhesion molecules Conjugation requires cell to cell contact between two cells of opposite mating type, usually the same species, During conjugation plasmid is replicated and single stranded copy is transferred to recipient. Recipient synthesizes complementary strand to complete plasmid plasmid can remain as separate circle or -plasmid can be integrated into host cell genome Transformation (genetics) From Wikipedia, the free encyclopedia Not to be confused with an unrelated process called malignant transformation which occurs in the progression of cancer. In this image, a gene from bacterial cell 1 is moved from bacterial cell 1 to bacterial cell 2. This process of bacterial cell 2 taking up new genetic material is called transformation. In molecular a cell resulting biology, transformation is from the direct uptake the genetic alteration and incorporation of of exogenousgenetic material (exogenous DNA) from its surroundings through the cell membrane(s). Transformation occurs naturally in some species of bacteria, but in biotechnology transformation is a basic technique, and can be made to occur in many kinds of cells. For transformation to happen in nature, bacteria must be in a state وفاء عبد الواحد.د المحاضرة االولى والثانية of competence, which might occur as a time-limited response to environmental conditions such as starvation and cell density. Transformation is one of three processes by which exogenous genetic material may be introduced into a bacterial cell, the other two beingconjugation (transfer of genetic material between two bacterial cells in direct contact) and transduction (injection of foreign DNA by abacteriophage virus into the host bacterium). "Transformation" may also be used to describe the insertion of new genetic material into nonbacterial cells, including animal and plant cells; however, because "transformation" has a special meaning in relation to animal cells, indicating progression to a cancerous state, the term should be avoided for animal cells when describing introduction of exogenous genetic material. Introduction of foreign DNA intoeukaryotic cells is often called "transfection".[1] resulting in permanent chromosomal ch Transduction Transduction is the process by which DNA is transferred from one bacterium to another by a virus . It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector. Transduction does not require physical contact between the cell donating the DNA and the cell receiving the DNA (which occurs in conjugation), and it is DNAase resistant (transformation is susceptible to DNAase). Transduction is a common tool used by molecular biologists to stably introduce a foreign gene into a host cell's genome. وفاء عبد الواحد.د المحاضرة االولى والثانية Transduction Transduction is the process by which DNA is transferred from one bacterium to another by a virus. It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector. When bacteriophages (viruses that infect bacteria) infect a bacterial cell, their normal mode of reproduction is to harness the replicational, transcriptional, and translation machinery of the host bacterial cell to make numerous virions, or complete viral particles, including the viral DNA or RNA and the protein coat. Transduction is especially important because it explains one mechanism by which antibiotic drugs become ineffective due to the transfer of antibiotic-resistance genes between bacteria. In addition, hopes to create medical methods of genetic modification of diseases such as Duchenne/Becker Muscular Dystrophy are based on these methodologies. The Lytic Cycle and the Lysogenic Cycle Transduction happens through either the lytic cycle or the lysogenic cycle. If the lysogenic cycle is adopted, the phage chromosome is integrated (by covalent bonds) into the bacterial chromosome, where it can remain dormant for thousands of generations. If the lysogen is induced (by UV light for example), the phage genome is excised from the bacterial chromosome and initiates the lytic cycle, which culminates in lysis of the cell and the release of phage particles. The lytic cycle leads to the production of new phage particles which are released by lysis of the host. Transduction is a method for transferring genetic material. The packaging of bacteriophage DNA has low fidelity and small pieces of bacterial وفاء عبد الواحد.د المحاضرة االولى والثانية DNA, together with the bacteriophage genome, may become packaged into the bacteriophage genome. At the same time, some phage genes are left behind in the bacterial chromosome. There are generally three types of recombination events that can lead to this incorporation of bacterial DNA into the viral D Transduction is especially important because it explains one mechanism by which antibiotic drugs become ineffective due to the transfer of antibiotic-resistance genes between bacteria. In addition, hopes to create medical methods of genetic modification of diseases such as Duchenne/Becker Muscular Dystrophy are based on these methodologies. The Lytic Cycle and the Lysogenic Cycle Transduction happens through either the lytic cycle or the lysogenic cycle. If the lysogenic cycle is adopted, the phage chromosome is integrated (by covalent bonds) into the bacterial chromosome, where it can remain dormant for thousands of generations. If the lysogen is induced (by UV light for example), the phage genome is excised from the bacterial chromosome and initiates the lytic cycle, which culminates in lysis of the cell and the release of phage particles. The lytic cycle leads to the production of new phage particles which are released by lysis of the host. Transduction is a method for transferring genetic material. The packaging of bacteriophage DNA has low fidelity and small pieces of bacterial DNA, together with the bacteriophage genome, may become packaged into the bacteriophage genome. At the same time, some phage genes are left behind in the bacterial chromosome. وفاء عبد الواحد.د المحاضرة االولى والثانية There are generally three types of recombination events that can lead to this incorporation of bacterial DNA into the viral DNA, leading to two modes of recombination. Generalized transduction is the process by which any bacterial gene may be transferred to another bacterium via a bacteriophage, and typically carries only bacterial DNA and no viral DNA. In essence, this is the packaging of bacterial DNA into a viral envelope. This may occur in two main ways, recombination and headful packaging. If bacteriophages undertake the lytic cycle of infection upon entering a bacterium, the virus will take control of the cell's machinery for use in replicating its own viral DNA. If by chance bacterial chromosomal DNA is inserted into the viral capsid which is usually used to encapsulate the viral DNA, the mistake will lead to generalized transduction. If the virus replicates using "headful packaging," it attempts to fill th Fates of DNA Inserted into the Recipient Cell When the new DNA is inserted into this recipient cell it can fall to one of three fates: the DNA will be absorbed by the cell and be recycled for spare parts; if the DNA was originally a plasmid, it will recirculate inside the new cell and become a plasmid again; if the new DNA matches with a homologous region of the recipient cell's chromosome, it will exchange DNA material similar to the actions in conjugation. This type of recombination is random and the amount recombined depends on the size of the virus being used. Specialized transduction is the process by which a restricted set of bacterial genes are transferred to another bacterium. The genes that وفاء عبد الواحد.د المحاضرة االولى والثانية get transferred (donor genes) depend on where the phage genome is located on the chromosome. Specialized transduction occurs when the prophage excises imprecisely from the chromosome so that bacterial genes lying adjacent to the prophage are included in the excised DNA. The excised DNA is then packaged into a new virus particle, which can then deliver the DNA to a new bacterium, where the donor genes can be inserted into the recipient chromosome or remain in the cytoplasm, depending on the nature of the bacteriophage. When the partially encapsulated phage material infects another cell and becomes a "prophage" (is covalently bonded into the infected cell's chromosome), the partially coded prophage DNA is called a "heterogenote. " Example of specialized transduction is λ phages in Escherichia coli, which was discovered by Esther Lederberg. Genetics of prokaryotes and viruses[change | change source] The genetics of bacteria, archaea and viruses is a major field or research. Bacterial mostly divide by asexual cell division, but do have a kind of sex by horizontal gene transfer.Bacterial conjugation, transduction and transformation are their methods. In addition, the complete DNA sequence of many bacteria, archaea and viruses is now known. Although many bacteria were given generic and specific names, like Staphylococcus aureus, the whole idea of a species is rather meaningless for an organism which does not have sexes and crossing- وفاء عبد الواحد.د المحاضرة االولى والثانية over of chromosomes.[27] Instead, these organisms have strains, and that is how they are identified in the laboratory. Genes and development[change | change source] Gene expression[change | change source] Gene expression is the process by which the heritable information in a gene, the sequence of DNA base pairs, is made into a functional gene product, such as protein or RNA. The basic idea is that DNA is transcribed into RNA, which is then translated into proteins. Proteins make many of the structures and all the enzymes in a cell or organism. Several steps in the gene expression process may be modulated (tuned). This includes both the transcription and translation stages, and the final folded state of a protein. Gene regulation switches genes on and off, and so controls cell differentiation, and morphogenesis. Gene regulation may also serve as a basis for evolutionary change: control of the timing, location, and amount of gene expression can have a profound effect on the development of the organism. The expression of a gene may vary a lot in different tissues. This is called pleiotropism, a widespread phenomenon in genetics. Alternative splicing is a modern discovery of great importance. It is a process where from a single gene a large number of variant proteins can be assembled. One particularDrosophila gene (DSCAM) can be alternatively spliced into 38,000 different mRNA.[28] وفاء عبد الواحد.د المحاضرة االولى والثانية DN The structure of part of a DNA double helix Chemical structure of DNA. The phosphate groups are yellow, the deoxyribonucleicsugars are orange, and thenitrogen bases are green,purple,pink, and blue. Theatoms shown are: P=phosphorus O=oxygenN=nitrogen H=hydrogen وفاء عبد الواحد.د المحاضرة االولى والثانية DNA being copied DNA, short for deoxyribonucleic acid, is the molecule that contains the genetic code of organisms. This includes animals, plants, protists,archaea and bacteria. DNA is in each cell in the organism and tells cells what proteins to make. Mostly, these proteins are enzymes. DNA is inherited by children from their parents. This is why children share traits with their parents, such as skin, hair and eye color. The DNA in a person is a combination of the DNA from each of their parents. Part of an organism's DNA is "non-coding DNA" sequences. They do not code for protein sequences. Some noncoding DNA is transcribed intononcoding RNA molecules, such as transfer RNA, ribosomal RNA, and regulatory RNAs. Other sequences are not transcribed at all, or give rise to RNA of unknown function. The amount of non-coding DNA varies greatly among species. For example, over 98% of the human genome is non-coding DNA,[1] while only about 2% of a typical bacterial genome is non-coding DNA. Viruses use either DNA or RNA to infect organisms.[2] The genome replication of most DNA viruses takes place in the cell's nucleus, whereas RNA viruses usually replicate in the cytoplasm. Contents [hide] 1Structure of DNA o 1.1Nucleotides وفاء عبد الواحد.د المحاضرة االولى والثانية o 1.2Chromatin 2Copying DNA o 2.1Mutations 3Protein synthesis 4History of DNA research 5Related pages 6References 7Other websites Structure of DNA DNA has a double helix shape, which is like a ladder twisted into a spiral. Each step of the ladder is a pair of nucleotides. Nucleotides A nucleotide is a molecule made up of: deoxyribose, a kind of sugar with 5 carbon atoms, a phosphate group made of phosphorus and oxygen, and nitrogenous base DNA is made of four types of nucleotide: Adenine (A) Thymine (T) Cytosine (C) Guanine (G) The 'rungs' of the DNA ladder are each made of two bases, one base coming from each leg. The bases connect in the middle: 'A' only pairs with 'T', and 'C' only pairs with 'G'. The bases are held together by hydrogen bonds. وفاء عبد الواحد.د المحاضرة االولى والثانية Adenine (A) and thymine (T) can pair up because they make two hydrogen bonds, and cytosine (C) and guanine (G) pair up to make three hydrogen bonds. Although the bases are always in fixed pairs, the pairs can come in any order (A-T or T-A; similarly, C-G or G-C). This way, DNA can write 'codes' out of the 'letters' that are the bases. These codes contain the message that tells the cell what to do. Chromatin On chromosomes, the DNA is bound up with proteins called histones to form chromatin. This association takes part in epigenetics and gene regulation. Genes are switched on and off during development and cell activity, and this regulation is the basis of most of the activity which takes place in cells. Copying DNA When DNA is copied this is called DNA replication. Briefly, the hydrogen bonds holding together paired bases are broken and the molecule is split in half: the legs of the ladder are separated. This gives two single strands. New strands are formed by matching the bases (A with T and G with C) to make the missing strands. First, an enzyme called DNA helicase splits the DNA down the middle by breaking the hydrogen bonds. Then after the DNA molecule is in two separate pieces, another molecule called DNA polymerase makes a new strand that matches each of the strands of the split DNA molecule. Each copy of a DNA molecule is made of half of the original (starting) molecule and half of new bases. Mutations وفاء عبد الواحد.د المحاضرة االولى والثانية When DNA is copied, mistakes are sometimes made – these are called mutations. There are three main types of mutations: Deletion, where one or more bases are left out. Substitution, where one or more bases are substituted for another base in the sequence. Insertion, where one or more extra base is put in. Duplication, where a sequence of bases pairs are repeated. Mutations may also be classified by their effect on the structure and function of proteins, or their effect on fitness. Mutations may be bad for the organism, or neutral, or of benefit. Sometimes mutations are fatal for the organism – the protein made by the new DNA does not work at all, and this causes the embryo to die. On the other hand, evolution is moved forward by mutations, when the new version of the protein works better for the organism. Protein synthesis A section of DNA that contains instructions to make a protein is called a gene. Each gene has the sequence for at least one polypeptide.[3] Proteins form structures, and also form enzymes. The enzymes do most of the work in cells. Proteins are made out of smaller polypeptides, which are formed of amino acids. To make a protein to do a particular job, the correct amino acids have to be joined up in the correct order. Proteins are made by tiny machines in the cell called ribosomes. Ribosomes are in the main body of the cell, but DNA is only in the nucleus of the cell. The codon is part of the DNA, but DNA never leaves the nucleus. Because DNA cannot leave the nucleus, the cell makes a وفاء عبد الواحد.د المحاضرة االولى والثانية copy of the DNA sequence in RNA. This is smaller and can get through the holes – pores – in the membrane of the nucleus and out into the cell. Genes encoded in DNA are transcribed into messenger RNA (mRNA) by proteins such as RNA polymerase. Mature mRNA is then used as a template for protein synthesis by the ribosome. Ribosomes read codons, 'words' made of three base pairs that tell the ribosome which amino acid to add. The ribosome scans along an mRNA, reading the code while it makes protein. Another RNA called tRNA helps match the right amino acid to each codon وفاء عبد الواحد.د المحاضرة االولى والثانية History of DNA research James D. Watson and Francis Crick (right), with Maclyn McCarty (left) DNA was first isolated (extracted from cells) by Swiss physician Friedrich Miescher in 1869, when he was working on bacteria from the pus in surgical bandages. The molecule was found in the nucleus of the cells and so he called it nuclein.[4] In 1928, Frederick Griffith discovered that traits of the "smooth" form of Pneumococcus could be transferred to the "rough" form of the same bacteria by mixing killed "smooth" bacteria with the live "rough" form.[5] This system provided the first clear suggestion that DNA carries genetic information. The Avery–MacLeod–McCarty experiment identified DNA as the transforming principle in 1943.[6][7] DNA's role in heredity was confirmed in 1952, when Alfred Hershey and Martha Chase in the Hershey–Chase experiment showed that DNA is the genetic material of the T2 bacteriophage.[8] وفاء عبد الواحد.د المحاضرة االولى والثانية In the 1950s, Erwin Chargaff [9] found that the amount of thymine (T) present in a molecule of DNA was about equal to the amount of adenine (A) present. He found that the same applies to guanine (G) and cytosine (C). Chargaff's rules summarises this finding. In 1953, James D. Watson and Francis Crick suggested what is now accepted as the first correct double-helix model of DNA structure in the journal Nature.[10] Their double-helix, molecular model of DNA was then based on a single X-ray diffraction image "Photo 51", taken by Rosalind Franklin and Raymond Gosling in May 1952.[11] Experimental evidence supporting the Watson and Crick model was published in a series of five articles in the same issue of Nature.[12] Of these, Franklin and Gosling's paper was the first publication of their own X-ray diffraction data and original analysis method that partly supported the Watson and Crick model;[13] this issue also contained an article on DNA structure by Maurice Wilkins and two of his colleagues, whose analysis and in vivo B-DNA X-ray patterns also supported the presence in vivo of the double-helical DNA configurations as proposed by Crick and Watson for their double-helix molecular model of DNA in the previous two pages of Nature. In 1962, after Franklin's death, Watson, Crick, and Wilkins jointly received the Nobel Prize in Physiology or Medicine.[14] Nobel Prizes were awarded only to living recipients at the time. A debate continues about who should receive credit for the discovery.[15] In 1957, Crick explained the relationship between DNA, RNA, and proteins, in the central dogma of molecular biology.[16] How DNA was copied (the replication mechanism) came in 1958 through the Meselson–Stahl experiment.[17] More work by Crick and coworkers showed that the genetic code was based on non-overlapping triplets of وفاء عبد الواحد.د المحاضرة االولى والثانية bases, called codons.[18] These findings represent the birth of molecular biology. How Watson and Crick got Franklin's results has been much debated. Crick, Watson and Maurice Wilkins were awarded the Nobel Prize in 1962 for their work on DNA – Rosalind Franklin had died in 1958. DNA replication From Wikipedia, the free encyclopedia DNA replication. The double helix is unwound and each strand acts as a template (blue) for the next strand. Bases are matched to synthesize the new partner strands (green). In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. This process occurs in all living organisms and is the basis for biological inheritance. DNA is made up of a double helix of two complementary strands. During replication, these strands are separated. Each strand of the original DNA molecule then serves as a وفاء عبد الواحد.د المحاضرة االولى والثانية template for the production of its counterpart, a process referred to as semiconservative replication. Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA replication.[1][2] In a cell, DNA replication begins at specific locations, or origins of replication, in the genome.[3] Unwinding of DNA at the origin and synthesis of new strands results in replication forks growing bidirectionally from the origin. A number of proteins are associated with the replication fork to help in the initiation and continuation of DNA synthesis. Most prominently, DNA polymerase synthesizes the new strands by addingnucleotides that complement each (template) strand. DNA replication occurs during the S-stage of interphase. DNA replication can also be performed in vitro (artificially, outside a cell). DNA polymerases isolated from cells and artificial DNA primers can be used to initiate DNA synthesis at known sequences in a template DNA molecule. The polymerase chain reaction (PCR), a common laboratory technique, cyclically applies such artificial synthesis to amplify a specific target DNA fragment from a pool of DNA. DNA structures[edit] DNA usually exists as a double-stranded structure, with both strands coiled together to form the characteristic double-helix. Each single strand of DNA is a chain of four types ofnucleotides. Nucleotides in DNA contain a deoxyribose sugar, a phosphate, and a nucleobase. The four types of nucleotide correspond to the four nucleobases adenine,cytosine, guanine, and thymine, commonly abbreviated as A,C, G and T. Adenine and guanine are purine bases, وفاء عبد الواحد.د المحاضرة االولى والثانية while cytosine and thymine are pyrimidines. These nucleotides form phosphodiester bonds, creating the phosphate-deoxyribose backbone of the DNA double helix with the nuclei bases pointing inward (i.e., toward the opposing strand). Nucleotides (bases) are matched between strands through hydrogen bonds to form base pairs. Adenine pairs with thymine (two hydrogen bonds), and guanine pairs with cytosine (stronger: three hydrogen bonds). DNA strands have a directionality, and the different ends of a single strand are called the "3' (three-prime) end" and the "5' (five-prime) end". By convention, if the base sequence of a single strand of DNA is given, the left end of the sequence is the 5' end, while the right end of the sequence is the 3' end. The strands of the double helix are anti-parallel with one being 5' to 3', and the opposite strand 3' to 5'. These terms refer to the carbon atom in deoxyribose to which the next phosphate in the chain attaches. Directionality has consequences in DNA synthesis, because DNA polymerase can synthesize DNA in only one direction by adding nucleotides to the 3' end of a DNA strand. The pairing of complementary bases in DNA (through hydrogen bonding) means that the information contained within each strand is redundant. Phosphodiester (intra-strand) bonds are stronger than hydrogen (interstrand) bonds. This allows the strands to be separated from one another. The nucleotides on a single strand can therefore be used to reconstruct nucleotides on a newly synthesized partner strand.[4] DNA polymerase[edit] Main article: DNA polymerase وفاء عبد الواحد.د المحاضرة االولى والثانية DNA polymerases adds nucleotides to the 3' end of a strand of DNA.[5] If a mismatch is accidentally incorporated, the polymerase is inhibited from further extension. Proofreading removes the mismatched nucleotide and extension continues. DNA polymerases are a family of enzymes that carry out all forms of DNA replication.[6] DNA polymerases in general cannot initiate synthesis of new strands, but can only extend an existing DNA or RNA strand paired with a template strand. To begin synthesis, a short fragment of RNA, called a primer, must be created and paired with the template DNA strand. DNA polymerase adds a new strand of DNA by extending the 3' end of an existing nucleotide chain, adding new nucleotides matched to the وفاء عبد الواحد.د المحاضرة االولى والثانية template strand one at a time via the creation of phosphodiester bonds. The energy for this process of DNA polymerization comes from hydrolysis of the high-energy phosphate (phosphoanhydride) bonds between the three phosphates attached to each unincorporated base. (Free bases with their attached phosphate groups are called nucleotides; in particular, bases with three attached phosphate groups are called nucleoside triphosphates.) When a nucleotide is being added to a growing DNA strand, the formation of a phosphodiester bond between the proximal phosphate of the nucleotide to the growing chain is accompanied by hydrolysis of a high-energy phosphate bond with release of the two distal phosphates as a pyrophosphate. Enzymatic hydrolysis of the resulting pyrophosphate into inorganic phosphate consumes a second high-energy phosphate bond and renders the reaction effectively irreversible.[Note 1] In general, DNA polymerases are highly accurate, with an intrinsic error rate of less than one mistake for every 107 nucleotides added.[7] In addition, some DNA polymerases also have proofreading ability; they can remove nucleotides from the end of a growing strand in order to correct mismatched bases. Finally, post-replication mismatch repair mechanisms monitor the DNA for errors, being capable of distinguishing mismatches in the newly synthesized DNA strand from the original strand sequence. Together, these three discrimination steps enable replication fidelity of less than one mistake for every 109 nucleotides added.[7] The rate of DNA replication in a living cell was first measured as the rate of phage T4 DNA elongation in phage-infected E. coli.[8]During the period of exponential DNA increase at 37 °C, the rate was 749 nucleotides per second. The mutation rate per base pair per replication during phage T4 DNA synthesis is 1.7 per 108.[9] وفاء عبد الواحد.د المحاضرة االولى والثانية Replication process[edit] Main articles: Prokaryotic DNA replication and Eukaryotic DNA replication DNA replication, like all biological polymerization processes, proceeds in three enzymatically catalyzed and coordinated steps: initiation, elongation and termination. Initiation[edit] Role of initiators for initiation of DNA replication. Formation of pre-replication complex. For a cell to divide, it must first replicate its DNA.[10] This process is initiated at particular points in the DNA, known as "origins", which are targeted by initiator proteins.[3] In E. coli this protein is DnaA; in yeast, وفاء عبد الواحد.د المحاضرة االولى والثانية this is the origin recognition complex.[11] Sequences used by initiator proteins tend to be "AT-rich" (rich in adenine and thymine bases), because A-T base pairs have two hydrogen bonds (rather than the three formed in a C-G pair) and thus are easier to decouple.[12] Once the origin has been located, these initiators recruit other proteins and form the prereplication complex, which unzips the double-stranded DNA. Elongation[edit] DNA polymerase has 5'-3' activity. All known DNA replication systems require a free 3' hydroxyl group before synthesis can be initiated (Important note: the DNA template is read in 3' to 5' direction whereas a new strand is synthesized in the 5' to 3' direction—this is often confused). Four distinct mechanisms for initiation of synthesis are recognized: 1. All cellular life forms and many DNA viruses, phages and plasmids use a primase to synthesize a short RNA primer with a free 3' OH group which is subsequently elongated by a DNA polymerase. 2. The retroelements (including retroviruses) employ a transfer RNA that primes DNA replication by providing a free 3′ OH that is used for elongation by the reverse transcriptase. 3. In the adenoviruses and the φ29 family of bacteriophages, the 3' OH group is provided by the side chain of an amino acid of the genome attached protein (the terminal protein) to which nucleotides are added by the DNA polymerase to form a new strand. 4. In the single stranded DNA viruses — a group that includes the circoviruses, the geminiviruses, the parvoviruses and others — and also the many phages and plasmids that use the rolling circle وفاء عبد الواحد.د المحاضرة االولى والثانية replication (RCR) mechanism, the RCR endonuclease creates a nick in the genome strand (single stranded viruses) or one of the DNA strands (plasmids). The 5′ end of the nicked strand is transferred to atyrosine residue on the nuclease and the free 3′ OH group is then used by the DNA polymerase to synthesize the new strand. The first is the best known of these mechanisms and is used by the cellular organisms. In this mechanism, once the two strands are separated, primase adds RNA primers to the template strands. The leading strand receives one RNA primer while the lagging strand receives several. The leading strand is continuously extended from the primer by a DNA polymerase with high processivity, while the lagging strand is extended discontinuously from each primer forming Okazaki fragments. RNase removes the primer RNA fragments, and a low processivity DNA polymerase distinct from the replicative polymerase enters to fill the gaps. When this is complete, a single nick on the leading strand and several nicks on the lagging strand can be found. Ligase works to fill these nicks in, thus completing the newly replicated DNA molecule. The primase used in this process differs significantly between bacteria and archaea/eukaryotes. Bacteria use a primase belonging to the DnaG protein superfamily which contains a catalytic domain of the TOPRIM fold type.[13] The TOPRIM fold contains an α/β core with four conserved strands in a Rossmann-like topology. This structure is also found in the catalytic domains of topoisomerase Ia, topoisomerase II, the OLD-family nucleases and DNA repair proteins related to the RecR protein. وفاء عبد الواحد.د المحاضرة االولى والثانية The primase used by archaea and eukaryotes, in contrast, contains a highly derived version of the RNA recognition motif (RRM). This primase is structurally similar to many viral RNA-dependent RNA polymerases, reverse transcriptases, cyclic nucleotide generating cyclases and DNA polymerases of the A/B/Y families that are involved in DNA replication and repair. In eukaryotic replication, the primase forms a complex with Pol α.[14] Multiple DNA polymerases take on different roles in the DNA replication process. In E. coli, DNA Pol III is the polymerase enzyme primarily responsible for DNA replication. It assembles into a replication complex at the replication fork that exhibits extremely high processivity, remaining intact for the entire replication cycle. In contrast, DNA Pol I is the enzyme responsible for replacing RNA primers with DNA. DNA Pol I has a 5' to 3' exonuclease activity in addition to its polymerase activity, and uses its exonuclease activity to degrade the RNA primers ahead of it as it extends the DNA strand behind it, in a process called nick translation. Pol I is much less processive than Pol III because its primary function in DNA replication is to create many short DNA regions rather than a few very long regions. In eukaryotes, the low-processivity enzyme, Pol α, helps to initiate replication. The high-processivity extension enzymes are Pol δ and Pol ε. As DNA synthesis continues, the original DNA strands continue to unwind on each side of the bubble, forming a replication fork with two prongs. In bacteria, which have a single origin of replication on their circular chromosome, this process creates a "theta structure" (resembling the Greek letter theta: θ). In contrast, eukaryotes have longer linear chromosomes and initiate replication at multiple origins within these.>[15] Replication fork[edit] وفاء عبد الواحد.د المحاضرة االولى والثانية Scheme of the replication fork. a: template, b: leading strand, c: lagging strand, d: replication fork, e: primer, f:Okazaki fragments Many enzymes are involved in the DNA replication fork. The replication fork is a structure that forms within the nucleus during DNA replication. It is created by helicases, which break the hydrogen bonds holding the two DNA strands together. The resulting structure has two branching "prongs", each one made up of a single strand of DNA. These two strands serve as the template for the leading and lagging strands, which will be created as DNA polymerase matches complementary nucleotides to the templates; the templates may be properly referred to as the leading strand template and the lagging strand template. DNA is always synthesized in the 5' to 3' direction. Since the leading and lagging strand templates are oriented in opposite directions at the replication fork, a major issue is how to achieve synthesis of nascent وفاء عبد الواحد.د المحاضرة االولى والثانية (new) lagging strand DNA, whose direction of synthesis is opposite to the direction of the growing replication fork. Leading strand[edit] The leading strand is the strand of nascent DNA which is being synthesized in the same direction as the growing replication fork. A polymerase "reads" the leading strand template and adds complementary nucleotides to the nascent leading strand on a continuous basis. The polymerase involved in leading strand synthesis is DNA polymerase III (DNA Pol III) in prokaryotes.[16] In eukaryotes, leading strand synthesis is thought to be conducted by Pol ε; however, this view has recently been challenged, suggesting a role for Pol δ.[17] Lagging strand[edit] The lagging strand is the strand of nascent DNA whose direction of synthesis is opposite to the direction of the growing replication fork. Because of its orientation, replication of the lagging strand is more complicated as compared to that of the leading strand. As a consequence, the DNA polymerase on this strand is seen to "lag behind" the other strand. The lagging strand is synthesized in short, separated segments. On the lagging strand template, a primase "reads" the template DNA and initiates synthesis of a short complementary RNA primer. A DNA polymerase extends the primed segments, forming Okazaki fragments. The RNA primers are then removed and replaced with DNA, and the fragments of DNA are joined together by DNA ligase. DNA polymerase III (in prokaryotes) or Pol δ (in eukaryotes) is responsible for extension of the primers added during replication of the وفاء عبد الواحد.د المحاضرة االولى والثانية lagging strand. Primer removal is performed by DNA polymerase I (in prokaryotes) and Pol δ (in eukaryotes).[18] Eukaryotic primase is intrinsic to Pol α.[19] In eukaryotes, pol ε helps with repair during DNA replication. Dynamics at the replication fork[edit] The assembled human DNA clamp, a trimer of the protein PCNA. 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.[20] This build-up forms a torsional resistance that would eventually halt the progress of the replication fork. Topoisomerases are enzymes that temporarily break the strands of DNA, relieving the tension caused by unwinding the two strands of the DNA helix; topoisomerases (including DNA gyrase) achieve this by adding negative supercoils to the DNA helix.[21] Bare single-stranded DNA tends to fold back on itself forming secondary structures; these structures can interfere with the movement of DNA polymerase. To prevent this, single-strand binding proteins bind to the DNA until a second strand is synthesized, preventing secondary structure formation.[22] Clamp proteins form a sliding clamp around DNA, helping the DNA polymerase maintain contact with its template, thereby assisting with وفاء عبد الواحد.د المحاضرة االولى والثانية processivity. 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 that releases the DNA polymerase. Clamp-loading proteins are used to initially load the clamp, recognizing the junction between template and RNA primers.[2]:274-5 DNA replication proteins[edit] At the replication fork, many replication enzymes assemble on the DNA into a complex molecular machine called the replisome. The following is a list of major DNA replication enzymes that participate in the replisome:[23] Enzyme DNA Helicase Function in DNA replication Also known as helix destabilizing enzyme. Unwinds the DNA double helix at the Replication Fork. Builds a new duplex DNA strand by adding nucleotides in the 5' to 3' direction. Also performs DNA Polymerase proof-reading and error correction. There exist many different types of DNA Polymerase, each of which perform different functions in different types of cells. A protein which prevents elongating DNA DNA clamp polymerases from dissociating from the DNA parent strand. وفاء عبد الواحد.د المحاضرة االولى والثانية Single-Strand Binding (SSB) Proteins Topoisomerase DNA Gyrase DNA Ligase Bind to ssDNA and prevent the DNA double helix from re-annealing after DNA helicase unwinds it, thus maintaining the strand separation, and facilitating the synthesis of the nascent strand. Relaxes the DNA from its super-coiled nature. Relieves strain of unwinding by DNA helicase; this is a specific type of topoisomerase Re-anneals the semi-conservative strands and joins Okazaki Fragments of the lagging strand. Provides a starting point of RNA (or DNA) for DNA Primase polymerase to begin synthesis of the new DNA strand. Lengthens telomeric DNA by adding repetitive Telomerase nucleotide sequences to the ends of eukaryotic chromosomes. This allows germ cells and stem cells to avoid the Hayflick limit on cell division.[24] Replication machinery[edit] Replication machineries consist of factors involved in DNA replication and appearing on template ssDNAs. Replication machineries include primosotors are replication enzymes; DNA polymerase, DNA helicases, DNA clamps and DNA topoisomerases, and replication proteins; e.g. وفاء عبد الواحد.د المحاضرة االولى والثانية single-stranded DNA binding proteins (SSB). In the replication machineries these components coordinate. In most of the bacteria, all of the factors involved in DNA replication are located on replication forks and the complexes stay on the forks during DNA replication. These replication machineries are called replisomes or DNA replicase systems. These terms are generic terms for proteins located on replication forks. In eukaryotic and some bacterial cells the replisomes are not formed. Since replication machineries do not move relatively to template DNAs such as factories, they are called a replication factory.[25] In an alternative figure, DNA factories are similar to projectors and DNAs are like as cinematic films passing constantly into the projectors. In the replication factory model, after both DNA helicases for leading stands and lagging strands are loaded on the template DNAs, the helicases run along the DNAs into each other. The helicases remain associated for the remainder of replication process. Peter Meister et al. observed directly replication sites in budding yeast by monitoring green fluorescent protein(GFP)-tagged DNA polymerases α. They detected DNA replication of pairs of the tagged loci spaced apart symmetrically from a replication origin and found that the distance between the pairs decreased markedly by time.[26] This finding suggests that the mechanism of DNA replication goes with DNA factories. That is, couples of replication factories are loaded on replication origins and the factories associated with each other. Also, template DNAs move into the factories, which bring extrusion of the template ssDNAs and nascent DNAs. Meister’s finding is the first direct evidence of replication factory model. Subsequent research has shown that DNA helicases form dimers in many eukaryotic cells and bacterial replication machineries stay in single intranuclear location during DNA synthesis.[25] وفاء عبد الواحد.د المحاضرة االولى والثانية The replication factories perform disentanglement of sister chromatids. The disentanglement is essential for distributing the chromatids into daughter cells after DNA replication. Because sister chromatids after DNA replication hold each other by Cohesin rings, there is the only chance for the disentanglement in DNA replication. Fixing of replication machineries as replication factories can improve the success rate of DNA replication. If replication forks move freely in chromosomes, catenation of nuclei is aggravated and impedes mitotic segregation.[26] Termination[edit] 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 way. Because eukaryotes have linear chromosomes, DNA replication is unable to reach the very end of the chromosomes, but ends at the telomereregion of repetitive DNA close to the ends. This shortens the telomere of the daughter DNA strand. Shortening of the telomeres is a normal process in somatic cells. As a result, cells can only divide a certain number of times before the DNA loss prevents further division. (This is known as the Hayflick limit.) Within the germ cell line, which passes DNA to the next generation, telomerase extends the repetitive sequences of the telomere region to prevent degradation. Telomerase can become mistakenly active in somatic cells, sometimes leading to cancer formation. Increased telomerase activity is one of the hallmarks of cancer. Termination requires that the progress of the DNA replication fork must stop or be blocked. Termination at a specific locus, when it occurs, involves the interaction between two components: (1) a termination site sequence in the DNA, and (2) a protein which binds to this sequence to وفاء عبد الواحد.د المحاضرة االولى والثانية physically stop DNA replication. In various bacterial species, this is named the DNA replication terminus site-binding protein, or Ter protein. 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 regulates this process through the use of termination sequences that, 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.[27] Regulation[edit] The cell cycle of eukaryotic cells. Eukaryotes[edit] Within eukaryotes, DNA replication is controlled within the context of the cell cycle. As the cell grows and divides, it progresses through stages in the cell cycle; DNA replication takes place during the S phase (synthesis phase). The progress of the eukaryotic cell through the cycle is controlled by cell cycle checkpoints. Progression through checkpoints is controlled through complex interactions between various proteins, including cyclins and cyclin-dependent kinases.[28] Unlike bacteria, eukaryotic DNA replicates in the confines of the nucleus.[29] وفاء عبد الواحد.د المحاضرة االولى والثانية The G1/S checkpoint (or restriction checkpoint) regulates whether eukaryotic cells enter the process of DNA replication and subsequent division. Cells that do not proceed through this checkpoint remain in the G0 stage and do not replicate their DNA. Replication of chloroplast and mitochondrial genomes occurs independently of the cell cycle, through the process of D-loop replication. Replication focus[edit] In vertebrate cells, replication sites concentrate into positions called replication foci.[26] Replication sites can be detected by immunostaining daughter strands and replication enzymes and monitoring GFP-tagged replication factors. By these methods it is found that replication foci of varying size and positions appear in S phase of cell division and their number per nucleus is far smaller than the number of genomic replication forks. P. Heun et al.(2001) tracked GFP-tagged replication foci in budding yeast cells and revealed that replication origins move constantly in G1 and S phase and the dynamicsdecreased significantly in S phase.[26] Traditionally, replication sites were fixed on spatial structure of chromosomes by nuclear matrix or lamins. The Heun’s results denied the traditional concepts, budding yeasts don't have lamins, and support that replication origins self-assemble and form replication foci. By firing of replication origins, controlled spatially and temporally, the formation of replication foci is regulated. D. A. Jackson et al.(1998) revealed that neighboring origins fire simultaneously in mammalian cells.[26] Spatial juxtaposition of replication sites brings clustering of replication forks. The clustering do rescue of stalled replication forks and favors normal progress of replication forks. Progress of وفاء عبد الواحد.د المحاضرة االولى والثانية replication forks is inhibited by many factors; collision with proteins or with complexes binding strongly on DNA, deficiency of dNTPs, nicks on template DNAs and so on. If replication forks stall and the remaining sequences from the stalled forks are not replicated, the daughter strands have nick obtained un-replicated sites. The un-replicated sites on one parent's strand hold the other strand together but not daughter strands. Therefore, the resulting sister chromatids cannot separate from each other and cannot divide into 2 daughter cells. When neighboring origins fire and a fork from one origin is stalled, fork from other origin access on an opposite direction of the stalled fork and duplicate the un-replicated sites. As other mechanism of the rescue there is application of dormant replication origins that excess origins don't fire in normal DNA replication. Bacteria[edit] Dam methylates adenine of GATC sites after replication. وفاء عبد الواحد.د المحاضرة االولى والثانية Most bacteria do not go through a well-defined cell cycle but instead continuously copy their DNA; during rapid growth, this can result in the concurrent occurrence of multiple rounds of replication.[30] In E. coli, the best-characterized bacteria, DNA replication is regulated through several mechanisms, including: the hemimethylation and sequestering of the origin sequence, the ratio of adenosine triphosphate (ATP) toadenosine diphosphate (ADP), and the levels of protein DnaA. All these control the binding of initiator proteins to the origin sequences. Because E. coli methylates GATC DNA sequences, DNA synthesis results in hemimethylated sequences. This hemimethylated DNA is recognized by the 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.[31] 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 DnaA-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. Polymerase chain reaction[edit] Main article: Polymerase chain reaction Researchers commonly replicate DNA in vitro using the polymerase chain reaction (PCR). PCR uses a pair of primers to span a target region in template DNA, and then polymerizes partner strands in each direction وفاء عبد الواحد.د المحاضرة االولى والثانية from these primers using a thermostable DNA polymerase. Repeating this process through multiple cycles amplifies the targeted DNA region. 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 annealing of new primers, and the polymerase extends from these. As a result, the number of copies of the target region doubles each round, increasing exponentially.[32] Notes[edit] 1. Jump up^ The energetics of this process may also help 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. The problem is that if the high energy triphosphates were on the growing strand and not on the free nucleotides, proof-reading by removing a mismatched terminal nucleotide would be problematic: Once a nucleotide is added, the triphosphate is lost and a single phosphate remains on the backbone between the new nucleotide and the rest of the strand. If the added nucleotide were mismatched, removal would result in a DNA strand terminated by a monophosphate at the end of the "growing strand" rather than a high energy triphosphate. So strand would be stuck and wouldn't be able to grow anymore. In actuality, the high energy triphosphates hydrolyzed at each step originate from the free nucleotides, not the polymerized strand, so this issue does not exist. References[edit] وفاء عبد الواحد.د المحاضرة االولى والثانية 1. Jump up^ Imperfect DNA replication results in mutations. Berg JM, Tymoczko JL, Stryer L, Clarke ND (2002). "Chapter 27: DNA Replication, Recombination, and Repair". Biochemistry. W.H. Freeman and Company. ISBN 0-7167-3051-0. External link in |chapter= (help) 2. ^ Jump up to:a b Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002). "5DNA Replication, Repair, and Recombination". Molecular Biology of the Cell. Garland Science. ISBN 0-8153-3218-1. External link in |chapter= (help) 3. ^ Jump up to:a b Berg JM, Tymoczko JL, Stryer L, Clarke ND (2002). "Chapter 27, Section 4: DNA Replication of Both Strands Proceeds Rapidly from Specific Start Sites". Biochemistry. W.H. Freeman and Company. ISBN 0-7167-3051-0. External link in |chapter= (help) 4. Jump up^ Alberts, B., et al., Molecular Biology of the Cell, Garland Science, 4th ed., 2002, pp. 238–240 ISBN 0-81533218-1 5. Jump up^ Allison, Lizabeth A. Fundamental Molecular Biology. Blackwell Publishing. 2007. p.112ISBN 978-1-4051-0379-4 6. Jump up^ Berg JM, Tymoczko JL, Stryer L, Clarke ND (2002). Biochemistry. W.H. Freeman and Company. ISBN 0-71673051-0. Chapter 27, Section 2: DNA Polymerases Require a Template and a Primer 7. ^ Jump up to:a b McCulloch, Scott D; Kunkel, Thomas A (January 2008). "The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases". Cell Research. 18 (1): 148– 161. doi:10.1038/cr.2008.4. PMC 3639319 . PMID 18166979. وفاء عبد الواحد.د المحاضرة االولى والثانية 8. Jump up^ McCarthy, David; Minner, Charles; Bernstein, Harris; Bernstein, Carol (October 1976). "DNA elongation rates and growing point distributions of wild-type phage T4 and a DNAdelay amber mutant". Journal of Molecular Biology. 106 (4): 963– 981. doi:10.1016/0022-2836(76)90346-6. PMID 789903. 9. Jump up^ Drake JW (1970) The Molecular Basis of Mutation. Holden-Day, San Francisco ISBN 0816224501 ISBN 978-0816224500 10.Jump up^ 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 11.Jump up^ Weigel C, Schmidt A, Rückert B, Lurz R, Messer W (November 1997). "DnaA protein binding to individual DnaA boxes in the Escherichia coli replication origin, oriC". The EMBO Journal. 16 (21): 6574– 83. doi:10.1093/emboj/16.21.6574. PMC 1170261 .PMID 9351837. 12.Jump up^ 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 13.Jump up^ Aravind, L.; Leipe, D. D.; Koonin, E. V. (1998). "Toprim—a conserved catalytic domain in type IA and II topoisomerases, DnaG-type primases, OLD family nucleases and RecR proteins". Nucleic Acids Research. 26 (18): 4205– وفاء عبد الواحد.د المحاضرة االولى والثانية 4213. doi:10.1093/nar/26.18.4205.PMC 147817 . PMID 9722641. 14.Jump up^ Frick, David; Richardson, Charles (July 2001). "DNA Primases". Annual Review of Biochemistry. 70: 39– 80. doi:10.1146/annurev.biochem.70.1.39. PMID 11395402. 15.Jump up^ Huberman, Joel A.; Riggs, Arthur D. (March 1968). "On the mechanism of DNA replication in mammalian chromosomes". Journal of Molecular Biology. 32 (2): 327– 341.doi:10.1016/0022-2836(68)90013-2. PMID 5689363. 16.Jump up^ Johnson, RE; Klassen, R; Prakash, L; Prakash, S (July 2015). "A Major Role of DNA Polymerase δ in Replication of Both the Leading and Lagging DNA Strands.". Molecular Cell. 59 (2): 163–175. doi:10.1016/j.molcel.2015.05.038. PMC 4517859 .PMID 26145172. 17.Jump up^ Stillman, Bruce (July 2015). "Reconsidering DNA Polymerases at the Replication Fork in Eukaryotes". Molecular Cell. 59 (2): 139– 141. doi:10.1016/j.molcel.2015.07.004.PMC 4636199 . PMID 26186286. 18.Jump up^ Distinguishing the pathways of primer removal during Eukaryotic Okazaki fragment maturation Contributor Author Rossi, Marie Louise. Date Accessioned: 2009-02-23T17:05:09Z. 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"E. coli SeqA protein binds oriC in two different methylmodulated reactions appropriate to its roles in DNA replication initiation and origin sequestration".Cell. 82 (6): 927–936. doi:10.1016/0092-8674(95)902724. PMID 7553853. 32. Jump up^ Saiki, Randall; Gelfand, David H.; Stoffel, Susanne; Scharf, Stephen J.; Higuchi, Russell; Horn, Glenn T.; Mullis, Kary B.; Erlich, Henry A. (29 January 1988). "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". Science.239 (4839): 487– 491. doi:10.1126/science.2448875. PMID 2448875. Retrieved 7 April2016. وفاء عبد الواحد.د المحاضرة االولى والثانية 2. Eukaryotes vs Prokaryotes 3. There is much conservation between the two systems, in as much as the enzymology, the replication fork geometry, the basic fundamental features and the use of multi-protein machinery are all very much the same in both. However, there are more protein components in the Eukaryotic replication machinery. In prokaryotes, the replication form moves 10x faster than in eukaryotes. Prokaryotic replication Eukaryotic replication semiconservative replication semiconservative replication single origin replication (oriC) multiple origins of replication (ARS) primer synthesized by primase primer synthesized by subunits of DNA polymerase α processing enzyme: DNA polymerase III processing enzymes: DNA polymerases α and δ removal of primer: DNA polymerase I removal of primer: DNA polymerase β DNA free in cytoplasm as nucleoid chromatin structure, chromosomes, histones circular DNA linear DNA: problem of replication of chromosome ends → telome