DNA replication in prokaryotes PDF

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DNA replication in prokaryotes (E. coli) DNA replication In molecular biology, DNA replication is the biological process of producing two identical copies of DNA from one original DNA molecule. In this process a single DNA molecules having two strands i.e the prenatal or old strands are used as template to produce daughter or new strands thus DNA can be replicated in a semi conservative method in which one strand are conserved. Now in this case we have one old and one new strand as shown in figure (1). Bacterial chromosomes or DNA can be replicated by theta mode of replication as shown in figure (2). P

roteins or enzymes involved during DNA replication in prokaryotes (E. coli) There are many proteins or enzymes used during DNA replication in prokaryotes (E. coli) Proteins or enzymes

Functions

1

Dna A protein

Unwind the DNA at ori C site.

2

Dna B protein (helicase)

Act as a helicase and breaks the H 2 bonds b/w the two strands to opened the replication fork.

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Dna C protein

Act as a loader and load or moves the Dna B protein to breaks the H2 bonds.

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Dna G protein (primase)

Synthesize the RNA primers that contain 5-10 RNA nucleotides.

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Dna gyrase (topoisomerase 2)

Relives the super coiling on DNA strand.

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Dna ligase

Ligate or joins the two segment or nicks among the strands.

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DNA polymerase 1

Filling the gaps by adding the correct DNA nucleotides after the RNA 1

primers are removed. 8

DNA polymerase 2

Involves during DNA repaire mechanisms

9

DNA polymerase 3

Add the DNA nucleotides in 5/-3/ direction

1 0

Ribonuclease (RNAse)

Removes the ribonucleotides from the RNA primers

11 Single stranded binding protein (SSB) 1 2

TUS protein

Prevents the reannealing of DNA strands. Involves in termination of DNA replication by halting the helicase.

Stages in DNA replication There are three stages or steps involves in DNA replication. 1. Initiation of DNA replication 2. Elongation of DNA replication 3. Termination of DNA replication

1. Initiation of DNA replication When the DNA molecules can start their replication process at specific region of DNA then it is called as initiation of DNA. The prokaryotes can start their DNA replication at a specific site of DNA molecules called as ori C (origins of replication). Ori C (origin of replication) In DNA molecules of prokaryotes there is a stretch of 3 consecutives of 13bp sequences called as 13mers like hexamers, tetramers that contain 13 bp i.e 5 / GATCTTTATTT 3/ / 3/ CTAGAAATAAA 5/ . These sequences are rich in adenine and thymine bases therefore it is also called AT rich region. This 13mers are also called DUE (duplex unwinding elements) because the DNA can unwind at that specific site.

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There are also a stretch of 4 sites (R boxes i.e R1-R4) which contain 9bp sequences called as 9mers which contain the bases like that 3/ TGTGAATAA 5/ / 5/ ACACTTATT 3/. There is IHF (integration host factor) protein site b/w the R1 and R5 boxes. Similarly there is FIS protein site b/w R2 and R4. When IHF protein bind at IHF site then it enhance the DNA replication initiation while the FIS protein that bind on FIS site can regulate the DNA replication. The R1, R2, and R4 are called Dna A boxes because Dna A protein can bind here because they have high affinity for binding. This 9mers sequences are also called DAR (DNA assembly region) because the DNA -ve supercoiling can occur here. These all 3 13mer and 4 9mers are called as ori C site as shown in figure (3). Process of initiation of DNA replication During DNA replication ATP energies are used to activate Dna A proteins. Then the 40 Dna A proteins can bind on 9mer sequences (R or Dna A boxes) in which the DNA are coiled around in each proteins and induce the topological stress that can cause the degeneration or unwinding of A-T rich region or 13mer sequences sites as shown in figure (4). Then the SSB proteins are activated and bind on single stranded DNA during unwinding. In order to separate the two strands completely by breaking the H 2 bonds b/w them the Dna B proteins is activated and make a complex with Dna C proteins called as Dna BC complex the Dna C can load the Dna B at DUE site. The Dna B then breaks the H 2 bonds b/w the bases in the DNA strands and we get the completely separated strand in the form of replication bubbles as shown in figure (5). After that Dna g or primase enzymes are activated and bind on the respective strands and place the RNA primers. Then DNA polymerase 3 is activated and binds to RNA primers and start the elongation process as shown in figure (6). 2. Elongation of DNA replication Elongation in DNA replication is the stage where the DNA polymerase 3 can binds on the primer and elongate the newly strand by synthesizing them i.e adding DNA nucleotides.

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Process of elongation of DNA replication Elongation will begin if there is RNA primer on the both strand after the formation of replication bubble. During elongation the DNA polymerase 3 can be used having two beta clamp i.e one for lagging strand and other for leading strand. DNA pol 3 have also many subunit i.e beta, alpha, gamma and epsilon. The DNA pol 3 is activated and loaded at the terminal side of the RNA primer on both strand i.e 3/ OH end. It should be noted that without RNA primer the DNA polymerase 3 cannot be able to start the elongation. Once the DNA polymerase 3 is loaded at the primer end having free 3/ OH group then it add the DNA nucleotide and elongate the newly synthesized strand only in 5/ to 3/ direction as shown in figure (7). Synthesis of leading and lagging strands As we know that one strand of the DNA molecule have 5/ to 3/ end while the other strand have 3/ to 5/ end then during elongation the upper strand will be continuously synthesized from 5 / to 3/ direction and we called that leading or continuous strand but the lower strand will be synthesize in a segment or in a fragment form and these fragment are called as Okazaki fragment because it was 1st discover by Okazaki and it should be synthesized in 3 / to 5/ direction. The RNA primers which are add again and again to the lower strand and due to which the lower strand is synthesized in discontinuous form due to 3/ to 5/ direction we called that lagging or discontinuous strand as shown in figure (8). Loop formation during elongation During elongation as we know that DNA Pol 3 can add nucleotide only in 5 / to 3/ direction but one strand (upper) should be synthesized in 5 / to 3/ direction having no issue but the other strand should be synthesized in 3/ to 5/ direction which can create a problem and complication because we have two strand should be synthesized in same direction and we have also 1 DNA Pol 3. So how is it possible that DNA Pol 3 can synthesize both leading and lagging strand in same direction? Now the bacterial cell can solve that problem by the formation of loop to the lower strand and due to this the DNA pol3 can get the same direction and elongate both strand 4

simultaneously but remember due to loop formation only the direction is 5 / to 3/ while the lower strand is synthesized in 3/ to 5/ direction because the DNA Pol 3 have two beta clamp for both leading and leaging strand. During the synthesis of the lower strand called leaging strand the DNA Pol3 can hold the primer at 3/ oh end and add the nucleotide until it reach to 5/ end. Once the DNA pol 3 reach to the 5/ end then it leave the stand hold another primer from 3 / oh end and the process is repeated again and again until till it reach to the ter site. During elongation as compare to the upper strand the lower strand can open so many times due to loop formation. In this way the enzymes can perform their function fastly and replication in prokaryotes are fastly as compare to eukaryotes. During elongation the RNA primers having RNA sequences should be removed by RNAse h or ribonuclease enzymes and the gaps which is formed during this way are filled by DNA pol 1 while the neak is sealed by DNA ligase and thus the elongation is completed (figure 9). 3. Termination of DNA replication During elongation when the nucleotides are add by DNA Pol 3 and the two replication bubbles having replication fork, Dna B protein and Dna gyrase meet at the point called ter/tus complex due to which the replication stopped is called termination of DNA replication. TUS/TER complex Now to explain the termination of DNA replication we have termination utilization site (TUS) protein that utilize the termination sequences (ter seq). The ter seq is 24 bp long genomes. The TUS protein and TER seq make a complex called TUS protein/TER sequence complex which are present at the opposite of Ori C site due to which replication are stopped because the ter sequences cannot stopped replication by itself and the two strands are separated by Topoisomerase 4. There are ten ter sites in which 5 (J, G, F, B, C) are present to the left side and 5 (A, D, E, I, H) are present to the right side of TUS/TER complex. DNA replication in prokaryotes are usually bidirectional it means that the two replication bubbles having replication fork, Dna B protein and DNA gyrase are moving in opposite direction simultaneously and meet at TUS/TER complex. The ter sites have two end i.e permissive and non permissive ends. The permissive end can carry on the replication by permitting the replication bubbles while the non permissive end can prevent the replication bubble to pass. At the right side of 5

TUS/TER complex the permissive end is present to the left and non permissive is to the right while at the left side of TUS/TER complex the permissive end is present to the right and non permissive is to the left. When the replication bubbles are moving and reach to the permissive site then it dissociate the TUS protein and the replication is carry on. When the two replication bubbles meet at TER/TUS complex then the TUS protein make a clip and prevent the Dna B protein to backword and when the Dna B protein reached at ter site of TER/TUS complex then the TUS protein flip the two or one bp i.e cytosine and guanine due to which the Dna B protein cannot be passed and the replication are stopped. The new strands which are formed are jumped and then separated when the topoisomerase 4 cleaved it (figure 10).

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