Topic – Replication of bacterial - PowerPoint Presentation

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Topic –Replication of bacterial chromosomePresented byPallavi MohiteAssistant ProfessorDepartment of BiotechnologyDeogiri College, Aurangabad

OutlineRequirementsMechanismPallavi MohiteAsst. Prof. DCAReplication of bacterial chromosome

Enzymes and proteins involved in DNA replicationPolymerase/ replicase:A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create two identical DNA duplexes from a single original DNA duplex.3 different polymerases are known in E coli and other prokaryotes.Pol I: Polymerase I is a DNA repair enzyme from the family A polymerases that has a 5’ to 3’ and 3’ to 5’ activity. Pol I accounts for more than 95% of polymerase activity in E. coli, although cells that lack this polymerase have been found and its activity can be replaced by the other four types of polymerase. This DNA polymerase has a poor processivity rate, adding around 15 to 20 nucleotides per second. Pol I begins the process of DNA elongation at a point called the “origin of replication” and about 400 base pairs downstream of this point, Pol III takes over replication, which it performs at a much higher speed.Pol II: Polymerase II is a DNA repair enzyme with a 3’ to 5’ exonuclease activity. Pol II is a family B polymerase and provides support to Pol III. When DNA acquires damage in the form of short gaps, which block Pol III activity, Pol II helps to remedy this problem by restarting DNA synthesis downstream of these gaps.

Pol III: This holoenzyme is the main polymerase in E.coli DNA replication and is one of the family C polymerases. Polymerase III is made up of the clamp-loading complex, the beta sliding clamp processivity factor and the Pol III core. The core comprises three subunits – the α subunit which is the polymerase activity hub, the δ subunit which is the exonucleolytic proofreader, and the θ subunit which may stabilize δ. The core and the beta sliding clamp are present in duplicate, to allow for processing of both the leading and lagging DNA strands.

DNA ligase: DNA ligase is an enzyme which can connect two strands of DNA together by forming a bond between the free 3’-OH and 5’- phosphate . It is used in cells to join together the Okazaki fragments which are formed on the lagging strand during DNA replication. It requires NAD+ as cofactor.

DNA helicase: DNA helicases are the ATP dependent enzyme that unwinds the DNA to promote the separation of strands of the DNA. DNA helicases unwind DNA at positions called origins where synthesis will be initiated. The process of breaking the hydrogen bonds between the nucleotide base pairs in double-stranded DNA requires energy. To break the bonds, helicases use the energy stored in a molecule called ATP, which serves as the energy currency of cells. DNA helicases also function in other cellular processes where double-stranded DNA must be separated, including DNA repair and transcription.

DNA gyrase: DNA gyrase is an essential bacterial enzyme that catalyzes the ATP-dependent negative super-coiling of double-stranded closed-circular DNA. Gyrase belongs to a class of enzymes known as topoisomerases that are involved in the control of topological transitions of DNA.it relaxes the positive supercoiling by nicking one of the strands and rotating it through the other strand. The nick is then resealed to reform the dsDNA.

Replisome : The replisome is a large protein complex that carries out DNA replication, starting at the replication origin. It contains several enzymatic activities, such as helicase, primase and DNA polymerase and creates a replication fork to duplicate both the leading and lagging strand.Primosome:  a primosome is a protein complex responsible for creating RNA primers on single stranded DNA during DNA replication. The primosome consists of proteins: DnaG primase, DnaB helicase.

OutlineIn molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule.It takes place during the interphase between two mitotic divisions.It’s a semiconservative process.It requiresA DNA template: parental strand on which the other new complementary strand is synthesized.A primer: basically RNA in natureDeoxyribonucleoside triphosphate (dATP,dTTP,dCTP,dGTP)DNA unwinding proteins: to unwind the double helix of DNA, also called as DNA binding proteinsRNA polymerase: for primer synthesisSuperhelix relaxing proteins : to relieve the strain imposed in the DNA double helix due to unwinding

Mg ++

DNA polymerase

: for synthesis of new DNA strand,

etc

R

eplication starts at a specific point called

origin of replication

, it starts with a

nick

or

incision

made by an enzyme

endonuclease

.

The two strands of DNA are unwind with the help of DNA unwinding proteins.Superhelix relaxing protein relieves the strain imposed in the DNA double helix due to unwinding.RNA primer initiates the replication process. RNA pol. Synthesize the RNA primer at the starting of DNA template close to the origin.Deoxyribose nucleotides are now added to the 3’ end of the primer, the main DNA strand is synthesized on the DNA template by DNA pol III.

DNA pol I degrades the RNA primer and catalyse the short DNA strand to replace the primer. The segment is joind by DNA ligase.Both the strands are replicated in 5’> 3’ direction, therefore…Replication takes place continuously on the leading strand, & discontinuously on the lagging strand. Short fragments are synthesized on the lagging strand called as ‘Okazaki Fragments’. These fragments are later joined by the enzyme DNA ligase.Replication may be unidirectional or bidirectional.

DNA replication takes place during interphase

MechanismE coli DNA synthesis genes:The overall process requires the product of the genes as follows:

Steps involved:3 stages of replication:InitiationElongationTermination

InitiationThe chromosomal DNA consists of multiple replication units or replicons. Replication is initiated at each replicon after a nick or break in one strand of parental DNA. Nick takes place at a specific point called initiation point, and is caused by incision enzyme(endonuclease).The site is called as Origin from where the actual replication starts.Role of membranes: the replication apparatus of E coli consists of various replicating enzymes, attached at the plasma membrane where DNA and plasma membrane are attached to each other. This point is known as replicating point. DNA thread moves through the replicating point and gets replicated.Initiation comprises of 3 steps:Recognition of origin seq. Opening of DNA duplex to generate a region of ssDNACapturing DNA B protein (5’ to 3’ helicase activity + primase activator)DNA A protein (initiator) + ATP complex binds to the 9 bp inverted repeats at the origin sequence . It promotes the opening of opening of DNA duplex at 13 bp inverted repeat seq. DNA B protein is transferred to the exposed ssDNA & causes unwinding of DNA in presence of ATP, SSB & DNA gyrase. This results in unwinding of the DNA duplex to form replication fork.

ElongationThis step requires DNA helicase, primase, DNA polymerase, SSB proteins, RNASe H, DNA ligase.Events in elongation: Helicase enzyme travels in 5’ to 3’ direction & generates the replication fork by opening the DNA duplex.DNA primase associates with DNA B helicase to form primosome complex. The complex synthesizes the multiple primers on the lagging template strand and single primer on leading template strand. RNA Primer is of 10 to 20 bp.Continuous synthesis on leading strandDNA pol III synthesizes the new daughter strand on the leading strand in continuation. DNA pol III recognises the RNA primer

Superhelix relaxing protein: in circular DNA untwisting imposes a strain. This strain is relieved by supertwisting in unreplicated part of the DNA. The reaction is carried out by Superhelix relaxing enzyme by nick- closing enzymatic activity.By introducing breaks in the non replicating region of DNA one strand is made to rotate upon the other, relieving the strain.the break is then closed by DNA ligase.Template DNA: template DNA acts as a master copy on which new DNA strand is synthesized. A complementary strand is synthesized according to the Watson n Crick base pairing.The newly synthesized strand has the same base pair composition as the parental DNA strands.

Helicase unwinding the DNA strand

Superhelix

relaxing proteins relieving the strain

RNA primer: initiation of DNA synthesis requires an RNA primer. DNA Pol. is unable carry out the de novo DNA synthesis. A pre existing polynucleotide chain is required to which polynucleotides are added. RNA primer is synthesized at the origin point on the DNA template by a modified E coli RNA pol. It is a short seq. with 5’ triphosphate end and 3’-OH end

Elongation :A new DNA strand is synthesized by addition of DNA nucleotides to the3’-OH of pre-existing RNA primer. Synthesis takes place in 5’ to 3’ direction by DNA pol. III. Now the newly synthesized DNA strands consists of RNA primer at 5’ end. The primer is hydrolyzed by 5’-> 3’ exonuclese activity of DNA pol I. Again the gap is filled by DNA nucleotides by DNA pol. I . Both the strands are joined by DNA ligase.Why RNA PRIMER???It has been suggested that laying down the first few nucleotides during DNA synthesis is subjected to error that the addition of nucleotides on the pre-existing polynucleotide chain. Hence any errors in the initial stages of synthesis are eliminated by formation of RNA primer first, then replacing it by another DNA strand. In addition, DNA polymerase enzymes cannot begin a new DNA chain from scratch.  They can only attach new nucleotides onto 3' OH group of a nucleotide in a preexisting strand.

Replicating forks: At the point where two strands are separated, a Y shaped replicating fork is formed. In a bidirectional replication the separated strands between the two forks appear as a bubble or an eye under electron microscope. In E coli there are generally 2 rep. forks and 1000s in eukaryotic chromosome.