DNA replication prokaryotes Class 3 2022.pdf

DNA replication (Prokaryotes) Replication of the Circular Bacterial DNA Figure 5-24 Molecular Biology of the Cell Bacterial Cell Division Ø Single, naked, circular DNA molecules Ø Located in an area of the cytoplasm called the nucleoid Ø cell division occurs by binary fission Ø first, the sing...

DNA replication (Prokaryotes) Replication of the Circular Bacterial DNA Figure 5-24 Molecular Biology of the Cell Bacterial Cell Division Ø Single, naked, circular DNA molecules Ø Located in an area of the cytoplasm called the nucleoid Ø cell division occurs by binary fission Ø first, the single circular DNA molecules replicates, producing two identical copies of the original Ø Next, the 2 DNA molecules move to opposite ends of the cell Ø Finally, the cytoplasm divides in half, producing 2 daughter cells, each of which have one copy of the original DNA molecule DNA Replication Forks Figure 5-6 and 5-7 Molecular Biology of the Cell Phosphodiester bond formation Figure 5-3 Molecular Biology of the Cell Net reaction for DNA replication n1dATP n2dGTP DNA n3dCTP Mg2+ n4dTTP Nucleotides n1dAMP n2dGMP n3dCMP + (n1+n2+n3+n4)ppi n4dTMP DNA Problem You have a 120 base pair template DNA. After one round of replication a) How many nucleotides will be incorporated into DNA? b) How many PPi molecules will be produced? Cell cycle in Eukaryotes and Prokaryotes Eukaryotes Prokaryotes C: Chromosome replication and segregation D: Division or cytokinesis DNA Replication in Slow-growing and Fast-growing Bacteria M. tuberculosis • DNA replication time 40 min • Cell doubling time 24 h E. coli • DNA replication time 40 min • Cell doubling time 30 min Different Phases of DNA Replication DNA Replication has 3 phases: •Initiation •Elongation •Termination Initiation phase -Starts at a precise place on the chromosome - OriC -Is Bidirectional -Two replication forks proceed in opposite directions around the chromosome -and meet near the trp locus DNA Replication in Bacteria Figure 5-25 Molecular Biology of the Cell Replication Origin Ø Replication proceeds bidirectionally from OriC OriC is made of 245 bp (highly conserved) Ø Initiator proteins (DNA A) (multiple molecules) bind OriC, causing localized strand separation Ø This allows DNA helicase to be recruited. Ø DnaB-DnaC complex acts as a helicase which functions to unwind DNA further. Ø This unwinding produces 2 replication forks. Ø This unwound single-strand region is kept single-stranded through the action of singlestrand binding proteins. Function of Primosome Ø The next step involves formation of a primosome with the attachment of the DnaG primase (RNA Polymerase) Ø Binds to the lagging strand of the template Ø The primosome has 6 other proteins: n, n’, n’’, i, DnaB and DnaC. The last two are DNA helicase Ø the primosome multiprotein complex synthesizes RNA oligonucleotides that prime DNA synthesis by DNA Poly III Elongation Phase – Chromosome Replication §The DNA is unwound ahead of the replication fork by DnaB helicase which is accompanied by DNA gyrase. §Two DNA polymerase III complexes are present. § Both polymerases can only polymerize nucleotides 5’ to 3’ § Thus, both strands must be synthesized in the 5’ to 3’ direction. § The copy of the parental 3’ to 5’ strand is synthesized continuously. It is the leading strand. § As the helix unwinds, the 5’ to 3’ strand is copied in a discontinuous fashion through synthesis of Okazaki fragments. This is the lagging strand. § Primers are removed, 3’ ends of the Okazaki fragments elongated and finally joined by DNA ligase DNA polymerases Ø DNA polymerases I, II, III Ø there are 3 DNA polymerases in E. coli Ø all use 4 dNTPS as substrates. eliminating ppi Ø all require a primer Ø DNA polymerase III is the polymerizing enzyme during chromosome replication Ø it requires a primer-template Ø all synthesize DNA 5’ ®3’ DNA polymerases also possess exonuclease activities Properties Pol I Pol II Pol III ________________________________________________ 5’-3’ polymerase yes yes yes 3’-5’ exonuclease yes yes yes 5’-3’ exonuclease yes no no § Pol III is the major enzyme for DNA replication § Pol I is the major enzyme for repair. It is also needed for RNA primer removal § Pol II is also involved in repair DNA Polymerase III – has two types of accessory proteins § clamp loading protein – loads polymerase onto the primer (at primer-template junction) § sliding clamp protein – binds to clamp loading protein forming a ring around the template. § The ring formed by sliding clamp maintains the association of the polymerase with the template Supercoiling of DNA Linking number, positive and negative supercoils Cooperative action of DNA helicase and Topoisomerase Function of DNA Gyrase Ø Unwinding of DNA by helicase is accompanied by DNA gyrase Ø DNA Gyrase introduces negative supercoils in the DNA (ATP dependent). Ø Inhibitors of DNA gyrase: novobiocin, coumermycin Nalidixic acid, quinolones (ciprofloxacin, norfloxacin) Bacterial Chromosome -single, circular, covalently closed double stranded DNA molecule -comprises 2% of cellular dry weight -molecular weight of 3 x 109daltons -no histone proteins 1300 µm in length (approx. 1mm) Negatively supercoiled Bacterial Nucleoid – DNA is organized into negatively supercoiled domains § Nucleoid – represents DNA tertiary structure. § Supercoiling occurs when circular Negatively supercoiled domain double-stranded DNA becomes twisted to that axis of helix is itself helical. § If overwound – twisted in direction of helix § If underwound – twisted in opposite direction of helix § Underwound DNA – forms negative supercoils § Overwound DNA – forms positive supercoils Arthur Kornberg Received Nobel Prize in 1959 for discovering DNA replication mechanism Replication Termination Ø E. coli has two primary sites - T1 and T2 Ø daughter DNA molecules are linked as concatemers - which are resolved by DNA gyrase Inhibitors of DNA Replication • actinomycin D, proflavine, ethidium bromide – intercalating agents • mitomycins – covalent crosslinking • metronidazole, nitrofurans – DNA strand breakage • DNA Gyrase inhibitors: • Novobiocin and coumermycin A1; produced from Strptomyces species, inhibit ATP binding • Nalidixic acid, Quinolone derivatives (ciprofloxacin), inhibits the resealing of the DNA strand, used for anthrax infections Point mutation in DNA-gyrase gene causes resistant to quinolone The bacteria will be resistant to Nalidixic acid Class Objectives DNA Replication (Prokaryotes) After this class students will know; u u u u u u u u u u u u u u The direction of DNA replication Chemical reaction of DNA replication Replication forks and replicon Leading and lagging strands Okazaki fragments Stages of replication process Replication origin Regulation of DNA replication initiation Role of DNA helicase Role of Primase differentiate functions of different DNA polymerases Proof reading function of DNA polymerases Essential role of DNA gyrase Replication inhibitors and their medicinal importance