1 8th and 9th lectures in molecular biology DNA REPLICATION -part 1 and 2By Sawsan sajid

2 DNA replication is the process of producing two identical copies from one original DNA molecule. This biological process occurs in all living organisms . It is the basis for biological inheritance. DNA is composed from two strands and each strand of the original DNA molecule serves as template for the production of the complementary strand, a process referred to as semiconservative replication and the process followed by proofreading or error-checking mechanisms to ensure correct reading to the genetic code,. like all biological polymerization processes(Transcription and Translation), the process involve 3 stages : 1- initiation 2-elongation and 3- termination. Early studies of DNA replication mainly depend on the observation of Meselson and Stahl(1958) ,the British biochemist John Carnis (1963) and the Japanese scientist Reiji Okazaki (1968)

3 Prokaryotic and eukaryotic DNA replication is bidirectionalThe experiment of John Cairns in 1963 demonstrated by autoradiography that the DNA of  Escherichia coli is a single circular not linear molecule that is replicated from a point or moving locus forming the ( replicating fork) at which both new DNA strands are being synthesized. The movement of this fork is bidirectional in another world there are two moving forks, traveling in opposite directions around the chromosome forming θ theta shape (the Greek letter) which look like a bubble . it start from one point called ori c (origin of replication) and replication continue till reaching the opposite direction in one point called Ter i( from terminus). Also the shape is called the (A-butter fly replication) (شكل يشبه حرف ثيتا الاغريقي او شكل الفراشه وهذا هو تضاعف كيرنس )

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5 Replication in eukaryotic cell start from more than one ori c(each 300bp there is oric ) so multiple replication bubbles will form thus replication here is faster than prokaryotic cell

6 DNA COULD BE SYNTHESISED IN LABThe dream become true for synthesizing part of the genome in lab after 3 decade from discovering DNA polymerase enzyme precisely in 1983 by Kary mullis who found a genious way to amplify (تكثير ) any part of the genomic DNA by PCR process (polymerase chain reaction)the process require the following:  Template DNA (genomic animal or plant cell , plasmid, cosmid, bacterial/yeast colony, etc.) primers :usually forward and reverse DNA primers(17-25bp) forwarded from 5 ́→ OH 3́ with free end thus DNA polymerase will use this end to add nucleotide to the newly formed strand .in nature this segment is synthesized by primase enzyme (RNA rather than DNA as will discussed latter) buffer for DNA polymerase enzyme To enhance enzyme activity we add MgCl2 or MgCl2 dNTPs :The four type is used (dATP, d TTP, d GTP, d CTP) . Taq DNA polymerase: heat stable enzyme is used here . Cos of its stability in heat during denarturation step (95Cº)

7 Polymerase chain reactionResearchers 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 from these primers using a thermostable Taq DNA polymerase. Repeating this process through multiple cycles produces amplification of 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

8 Properties of Taq DNA polymeraseTaq polymerase is a thermostable DNA polymerase named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated by Thomas D. Brock. It is often abbreviated to "Taq Pol" and is frequently used in polymerase chain reaction(PCR), a method for greatly amplifying short segments of DNA . T. aquaticus is a bacterium that lives in hot springs and hydrothermal vents, and Taq polymerase was identified as an enzyme able to withstand the protein-denaturing conditions (high temperature) required during PCR.Therefore it replaced the DNA polymerase from E. coli originally used in PCR.Taq's optimum temperature for activity is 75–80°C, with a half-life of greater than 2 hours at 92.5°C , and can replicate a 1000 base pair strand of DNA in less than 10 seconds at 72°C. One of Taq's drawbacks مساوئ is its relatively low replication fidelity دقة It lacks a 3' to 5' exonuclease proofreading activity, and has an error rate measured at about 1 in 9,000 nucleotides.[ The remaining two domains however may act in coordination, via coupled domain motion. Some thermostable DNA polymerases have been isolated from other thermophilic bacteria and archaea, such as vent and Pfu DNA polymerase, possessing a proofreading activity, and are being used instead of (or in combination with) Taq for high-fidelity amplification.

9 Enzymes involve in DNA replication

10 DNA Helicase Also known as helix destabilizing enzyme cases formation of Replication Fork due to broken hydrogen bonds DNA Polymerase Builds a new duplex DNA strand by adding nucleotides in the 5' to 3' direction. performs proof-reading and error correction. DNA clamp: A protein (unit from polymerase which prevents DNA polymerase III from dissociating from the DNA parent strand. Single-Strand Binding (SSB) Proteins Bind to ssDNA and prevent the DNA double helix from re-annealing after DNA helicase unwinds it thus maintaining the strand separation DNA Gyrase (and Topoisomerase IV) ; this is a specific type of topisomerase II convert relaxed form to super coiled DNA Ligase Re-anneals the semi-conservative strands and joins Okazak’i Fragments of the lagging strand. Primase Provides a starting point for DNA polymerase to begin synthesis of the new DNA strand. Topoisomerase I : Relaxes the DNA from its super-coiled nature Telomerase Lengthens telomeric DNA by adding repetitive nucleotide sequences to the ends of eukaryotic chromosomes

11 Some important and basic informationPrimase: in fact is RNA polymerase thus the formed primer is RNA rather than DNA and it will removed latter by DNA polymerase I Topoisomerase I: will break the 3́́ 5́ phosphodiester bond converting super coiled to relax form which opposite to ligase Helicase : will break hydrogen bond between the two strand Movement of replication fork 5́ →3́́ which is the same direction of polymerization and direction of Leading strand (الشريط القائد) While the direction of lagging strand(الشريط الخامل) is 3́́ →5́ Polymerization in leading strand is continuously but it is un continuously in lagging strand thus okazaki fragment will form Okazaki fragments are between 1,000 and 2,000 nucleotides  long in Escherichia coli and are between 100 and 200 nucleotides long in eukaryotes. They are separated by ~10-nucleotide RNA primers and are un ligated until RNA primers are removed, followed by enzyme ligase connecting (ligating) the two Okazaki fragments into one continuous newly synthesized complementary strand

12 FULL ANSWER DNA polymerase, an enzyme responsible for carrying out synthesis, adds nucleotides to an existing DNA strand in the opposite direction of that strand's orientation. Thus, a strand must be oriented 3' to 5' so the enzyme can add nucleotides in the 5' to 3' direction. When DNA replication begins, small portions of the DNA double helix unwind at various points along the molecule called replication origins. Each opening is called a replication fork and serves as a spot where each strand of the DNA molecule can become a newly replicated molecule through the addition of nucleotides. Because each new molecule features one old strand from a previous molecule and one newly synthesized strand, DNA replication is said to be semiconservative. The leading strand is oriented 3' to 5', meaning new nucleotides can readily be added in the opposite 5' to 3' direction without interruption. However, in the case of the lagging strand, which is oriented 5' to 3', DNA polymerase must add new nucleotides in the direction facing away from the replication fork. As replication continues, this fork continues to open more along the strand, so DNA polymerase must continually reorient itself, causing replication to occur in fragments. DNA polymerase has 5'-3' activity. All known DNA replication systems require a free 3' hydroxylgroup before synthesis can be initiated (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 DNA synthesis are recognized:

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14 Types of DNA polymerase in Prokaryotic cellTypes of enzyme Initiation activity Polymerization 5́́→3́ Exonuclease activity 3́→5́ Exonuclease activity 5́́→3́ DNA polymerase I - + DNA polymerase II DNA polymerase III

15 Types of DNA polymerase in Eukaryotic cellThe DNA polymerases of eukaryotes are in general less understood than the DNA polymerases of prokaryotes, Eukaryotic cells have at least five major nuclear DNA polymerases: αالفا ,βبيتا ,γ, كاما δ , دلتا εكاما . ابسلون is found in mitochondria, although it is encoded by a nuclear gene. Plant chloroplasts also containtheir own DNA polymerase that appears to be similar to γ Pol α polymerase: it is the only enzyme has primase activity beside DNA polymerase so it is self- primed it will form short primer nts called the initiator RNA iRNA) Pol β polymerase: excision repair and it is not highly active and is not very processive. Pol γ polymerase: polymerization the mitochondrial DNA beside reparing by its exonuclease activity 3́→5́ Polδ دلتا and ε ابسلون polymerase :polymerization lagging (δ)and leading (ε) strand respectively 5 ́→3́. In eukaryotes, the low-processivity initiating enzyme, Pol α, has intrinsic primase activity. The high-processivity extension enzymes are Pol δ and Pol ε.

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18 Differences between leading and lagging strand replication

19 A new DNA strand is always synthesized in a 5’ to 3’ manner, thus the replication of both the strands goes in two different ways. 1- Leading strand A leading strand is the strand which is run from 5’-3’direction or the direction the same as the replication fork movement. It is synthesized continuously; there are no breaks in-between. This strand is formed as nucleotides are continuously added to the 3’ end of the strand after polymerase reads the original DNA template . Only one primer will require here .no Okazaki fragment will formed. 2 DNA polymerase molecules are required for polymerization the two strands which run together in the same machine binding together but still the replication happened in opposite direction The polymerase involved in leading strand synthesis is DNA polymerase III(DNA Pol III) in prokaryotes and presumably Pol ε in yeasts. In human cells the leading and lagging strands are synthesized by Pol εابسلون and Pol δ , respectively, within the nucleus and Pol γ in the mitochondria.[

20 2- lagging strand: . A lagging strand is the strand which is orianted in the 3’-5’ direction or opposite direction as to the movement of the replication fork. It grows or is synthesized away from the fork. Its movement in the opposite direction is the cause why it is discontinuous; it is synthesized in fragments. The primase, which is responsible for adding an RNA primer, has to wait for the fork to open before putting in the primer. The lagging strands have fragments of DNA which are called Okazaki fragments. More than one primer will be necessary here and it will be removed latter by the exonuclease activity of DNA polymerase (I) which will fill the gap between two adjacent akazaki fragments .the final binding will done by the activity of ligase enzyme who will add a 3́ 5́ phospho diester bond continuously ; this is the reason why the synthesis of the lagging strand is more complicated than the leading strand.

21 The DNA replication machinery ماكنة تضاعف الدناThe Replisome is composed of the following: 2 DNA Pol III enzymes molecules , each has a core subunits composed from 3 sub units α, ε and θ subunits. the α subunit has the polymerase activity. the ε subunit has 3'→5' exonuclease activity. the θ subunit stimulates the ε subunit's proofreading. 2 β units which act as sliding  clamps(مثبت المتزحلق (keeping the polymerase bound to the DNA template . 2 τ units which acts to dimerism two of the core enzymes (α, ε, and θ subunits). The gamma  γ complex   which acts as a clamp loader (مثبت الحامل او المقود) for the lagging strand helping the two β subunits to form one unit and bind to DNA. The γ unit is made up of 5 subunits which include 3 γ subunits, 1 δ subunit , and 1 δ' subunit . The δ is involved in copying of the lagging strand. Beside that there are Χ  and Ψ  which complete the complex and bind to γ DNA polymerase III synthesizes base pairs at a rate of around 1000 nucleotides per second. DNA Pol III activity begins after strand separation at the origin of replication. Because DNA synthesis cannot start replication , an RNA primer, complementary to part of the single-stranded DNA, is synthesized by primase (an RNA polymerase)

22 Structure and sub units of 2 DNA polymerase III(replisome)

23 Steps of DNA replication1- Initiation : the process require replictor and intiator protein(DnaA protein ). For a cell to divide, it must first replicate its DNA.This process is initiated at particular points in the DNA, known as replicator ( bp) which contain specific area called "origin of replication ori c or Dna A box) ", which will opened by  initiator proteins. In E. coli this protein is called  DnaA protein ; in yeast, is called  origin recognition complex.  Sequences opened 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 bond in a C-G pair). Once the origin has been recognized ,the initiators proteins (DnaA protein )start forming the pre-replication complex, which unwind the double-stranded DNA .All known DNA replication systems require a free 3' hydroxyl group before synthesis can be initiated . use a primase enzyme (RNApolymerase) to synthesize a short RNA primer(10-20 bp) with a free 3′ OH group which is subsequently elongated by a DNA polymerase in this mechanism, In eukaryotes, primase is produce by Pol α DNA polymerase and Pol δ/Pol ε are responsible for extension of the primed segments

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26 Replication fork The replication fork is a structure that forms during DNA replication .Many enzymes are involved in the DNA replication fork in order to stabilize initiation step . 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 templates. SSBPs also required here 2- Elongation step 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 (new) lagging strand DNA, whose direction of synthesis is opposite to the direction of the growing replication fork. :

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28 1-The leading strand receives one RNA primer while the lagging strand receives several2- The leading strand is continuously extended from the primer by a high processivity (متنامي ,متقدم وعامل ), replicative DNA polymerase, while the lagging strand is extended discontinuously from each primer, forming Okazaki fragments As DNA synthesis continues, the original DNA strands continue to unwind on each side of the bubble, forming a replication fork with two prongs(شوكة) 3-β Clamp proteins : it  form a sliding clamp around DNA, helping the DNA polymerase maintain contact with its template, thereby assisting with 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 .

29 Lagging strand :it is synthesized in short, separated segmentsLagging strand :it 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 III extends the primed segments, forming Okazaki fragments.  DNA polymerase will add nucleotides in the 5' to 3' direction; however, one of the parent strands(lagging) of DNA is 3' to 5' while the other (leading) is 5' to 3'. To solve this problem, replication occurs in opposite directions. lagging strand run away from the replication fork, and synthesized a series of short fragments known as Okazaki fragments, consequently requiring many primers. The RNA primers of Okazaki fragments are degraded by Rnase H and DNA Polymerase I

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31 3-Termination 1-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 physically stop DNA replication. In various bacterial species, this is named the DNA replication terminus site-binding protein, or Ter protein. 2-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 . As a result, the replication forks are constrained to always meet within the termination region of the chromosome. 3-Removes the primer (RNA fragments), by DNA polymerase I by 5'-3' exonuclease activity of polymerase I, and replaces the RNA nucleotides with DNA nucleotides. and fill the gaps. 4- When this is complete, a single nick on the leading strand and several nicks on the lagging strand can be found. 5- Ligase works to fill these nicks in, thus completing the newly replicated DNA molecule . 6- Topoisomerase IV will : separate the two complete daughter chromosome in to two chromosome

32 Termination in Eukaryotic cellPrimer removal in eukaryotes is performed by RNase I that remove all the primer leaving only one nucleotide in the junction between 2 nucleotide and the remained one will removed by FenI enzyme . Eukaryote cell 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 Telomere region of repetitive DNA close to the end. 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. 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. in the end of the replication the DNA will warp around the basic hiatons to form the chromatin