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Bacterial Chromosome Replication, Structure and Segregation Francis Crick and James Watson Bacterial Chromosome Replication, Structure and Segregation Objectives 1. Develop a thorough understanding of DNA structure because it is fundamental to the comprehension of: a. gene replication b. gene expression c. gene regulation d. gene mutation and repair of DNA damage e. recombination f. genome packaging and transmission g. gene manipulation h. genomics 2. Develop a thorough understanding of the process of DNA replication. 3. Begin to comprehend the practical applications that have been derived from the basic knowledge of DNA structure and replication. Experiments that demonstrated that DNA is the genetic material of bacteria and at least some bacteriophage or Insights gained from transformation and virus infection The Griffith experiments Heat-killed pathogenic encapsulated bacteria can convert nonpathogenic noncapsulated bacteria to the pathogenic capsulated form. R indicates roughcolony formers. S indicates smoothcolony formers. Roman numerals indicate serotype of the capsid polysaccharide, a genetically determined trait. Bacterial type Effect on mouse A Bacteria recovered None Live type IIR Nonpathogenic B Type IIIS Live type IIIS Pathogenic C Heat-killed type IIIS None Nonpathogenic D Mixture of live type IIR and heat-killed type IIIS Type IIIS Pathogenic Figure 6.1 Streptococcus pneumoniae colonies on medium solidified with agar R strain S strain Colonies of rough (R, small colonies) and smooth (S, large colonies) strains of Streptococcus pneumoniae. The S colonies are larger because each cell has a very thick, gelatinous, polysaccharide capsule, which is missing in R mutants. Photograph reproduced from from O. T. Avery, C. M. MacLeod, and M. McCarthy. 1944. J. Experimental Medicine 79: 137. Avery, McLeod and McCarthy Experiment I Heat kill Type IIIS Type IIR Type IIIS Extract macromolecular components Polysaccharides Lipids Proteins Nucleic acids (DNA + RNA) TRANSFORM LIVE TYPE IIR CELLS ? IIR IIR IIR IIR + IIIS The transforming principle is a nucleic acid, NOT a polysaccharide Avery, McLeod and McCarthy Experiment II Type IIR Type IIIS Fig 2.3 in Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. DNA, rather than RNA, is the D. pneumoniae transforming principle Hershey-Chase experiment demonstrating that DNA is the genetic material of bacteriophage T2 Alfred Hershey and Martha Chase knew that T-even bacteriophage (T2, T4, T6) consist of protein and DNA. They also knew that phage proteins contain sulfur and no phosphate, and phage DNA contains phosphate but no sulfur. They decided that these characteristics could be used to determine experimentally if the genes of bacteriophages consist of protein or DNA. Hershey-Chase experiment demonstrating that DNA is the genetic material of bacteriophage T2 Hershey and Chase also knew that many progeny phage are produced and released after a phage infected a bacterial cell. NEXT Hershey-Chase experiment demonstrating that DNA is the genetic material of bacteriophage T2 Armed with all this knowledge, Hershey and Chase designed an experiment to determine if protein or DNA is the “genetic material”, i.e. the substance that is passed from one generation to the next. A. Preparation of the experimental material: radioactively-labeled T2 bacteriophage. NEXT Modified from Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing s Benjamin Cummings. B. Part of the Hershey and Chase experiment that showed that DNA is the genetic material of bacteriophage T2. Collect cells by centifugation to remove phage "ghosts" 32P 35 S EUREKA! DNA is transmitted from the parental phage to the progeny phage and protein is not! The secret is in the molecular structure of DNA! Erwin Chargaff did quantitative chemical analyses of of DNA from different organisms and observed that: [A] = [T] and [G] = [C], but [A+T] ≠ [G+C] "Pairing I used later, translating my word into what had become a slogan. I did not say they were in a double structure, no. That is Crick and Watson. The helix is a gimcrack. The fact that it is double is Erwin important because it is an automatic way of reproduction. I never Chargaff claimed it was my idea, and I don't wish to." Interview with Erwin Chargaff, OMNI, 7, no. 9 (June 1985): 132. June 1985. Rosalind Franklin Maurice Wilkins Concluded from X-ray diffraction data that DNA is a helix DNA X-ray diffraction pattern DNA A double helix with paired bases: potentially contains information for its own replication (base pairing) and can encode gene products such as enzymes (base sequence). James Watson and Francis Crick Used the data to develop a structure that fit the data DNA 2 nm (20 Å) 3’ 5’ Minor groove Major groove 2.2 nm Major groove 5’ 3’ Hydrogen bonds Deoxyribose-phosphate Backbones 1 turn = 3.4 nm = 10 base pairs Minor groove 1.2 nm Deoxyribose-phosphate Backbones Modified from Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. A B DNA Replication In 1953 Watson and Crick PREDICTED that DNA replication is semiconservative, meaning that each strand can act My Heroes as a template for the synthesis of its complementary strand: two new copies are made, each consisting of an “old” (template) strand and a “new” strand. 3’ 5’ A B In 1958, Matthew Messelson and Franklin Stahl confirmed experimentally that DNA replication is semiconservative. 3’ 5’ 3’ 5’ The Messelson Stahl Experiment In 1958, Matt Messelson and Frank Stahl designed an experiment to discriminate among these possibilities. Important features: 1. E. coli could grow in a medium having 15NH4Cl instead of the normal ammonium chloride. The heavy nitrogen eventually replaced the normal nitrogen in the cell’s molecules. If returned to the normal medium, the 14N isotope would subsequently be incorporated. 2. DNAs having the heavy or light isotopes of N have a different density and can be separated in a CsCl buoyant density gradient. 14N Hybrid 15N 1:1 3 : 1 The Messelson Stahl Experiment Question What would Messelson and Stahl have observed in the CsCl equilibrium gradients after one and two generations if DNA replication would have been A. Conservative: the parental strands remain together and the two daughter strands together are a replica of the parental dsDNA? B. Dispersive: both strands of the parental DNA are broken up and pieces, together with additional nucleotides, are assembled into two DNA molecules in which both strands have some parental and some new pieces? The pieces could be individual nucleotides. Components of nucleotides Fig.1.2 BASES Purines Purine Adenine Guanine Pyrimidines Pyrimidine Cytosine Uracil Thymine O Sugars (Pentoses) Na+ −O P O− +H O− K+ 2-Deoxyribose Ribose Phosphate Nucleotide and nucleosides DNA nucleotide dAMP RNA nucleotide UMP Nucleotides 5’ 5’ 5’ dGMP 3’ 3’ 3’ dCMP 9 5’ 3’ Fig.1.2 5’ dGTP Phosphodiester bonds and polarity of polynucleotides 5’ end 3’ end DIRECTION OF NUCLEOTIDE SEQUENCE 3’ 5’ upstream downstream site Oligonucleotides < 100 bases Polynucleotides > 100 bases Fig 1.3 Base pairing with hydrogen bonding Fig 1.4 Polarity and Antiparallel Orientation of Strands in DNA Bases can be flipped out of the double helix by DNA processing and repair enzymes. Antiparallel strands Base flipped DNA Base Flipping by Enzymes DNA modifying enzymes N-glycosylases (remove bases from pentose in DNA backbone) DNA repair endonucleases Model of DNA Double Helix Showing Potential Protein Contact Points the Major and Minor Grooves Phosphate-ribose backbones Proteins interact with DNA mainly by binding to atoms of bases exposed in the major groove (shown in red). Minor groove Major groove Modified from Fig 7.5 in Madigan and Martinko 2006. Brock Biology of Microorganisms. Architecture of Prokaryotic Chromosomes 1. Most of the prokaryotes have a single circular chromosome. 2. Some prokaryotes have two or three, perhaps even more, circular chromosomes. 3. Some prokaryotes have at least one linear chromosome of either the “invertron” or “hairpin telomere” type. 4. Organisms that have circular chromosomes tend to have circular plasmids, and organisms that have linear chromosomes tend to have linear plasmids. However, this is not a hard and fast rule: there can be disagreements! For example, Rhodococcus fasciens has a circular chromosome, but at least one of its multiple plasmids is linear. 5. The shape of the chromosome affects several aspects of chromosome replication and distribution. Some examples of bacterial genome organization Bacterium Escherichia coli K12 Bacillus subtilis Xylella fastidiosa Chromosome(s) one circular (4.6 Mb) one circular (4.2 Mb) one circular (2.7 Mb) Brucella melitensis Vibrio cholera Deinococcus radiodurans Rhizobacterium melliloti two two two two Paracoccus denitrificans three circular (2.0 + 1.1 + 0.64 Mb) Streptomyces coelicolor one linear (8.7-Mb invertron) one linear (356 Kb) + one circular (31 Kb) Borellia burgdorferi one linear (0.9-Mb hairpin) multiple linear and circular (9 - 54 Kb) circular circular circular circular (2.1 (2.9 (2.6 (3.4 + 1.2 Mb) +1.1 Mb) + 0.4 Mb) + 1.7 Mb) Plasmids two circular (51 + 1.3 Kb) two circular (177 + 45 Kb) one circular megaplasmid (1.4 Mb) Expanded from Ochman H. 2002. Bacterial evolution: chromosome arithmetic and geometry. Curr. Biol. 12:R427–28. Replication of Circular Chromosomes The E. coli Paradigm Proteins involved in E. coli DNA replication Protein Gene Function DnaA dnaA Initiator protein, primosome (priming complex) formation DnaB dnaB DNA helicase (strand separation; hexamer) DnaC dnaC Delivers DnaB to replication complex SSB Primase ssb dnaG Binding to single-stranded DNA RNA primer synthesis DNA Pol III 11 genes DNA replication (presented in next slide) DNA Pol I polA Primer removal, gap filling DNA ligase lig Sealing DNA nicks DNA gyrase α subunit gyrA DNA supercoiling (unwinding) Nick closing β subunit gyrB ATPase topA DNA supercoiling (DNA “packaging”) Topo I Pol III TABLE 1.1 The 11 polypeptides of the E. coli DNA Pol III (holoenzyme) Subunit Gene Function α ε RNase H dnaE dnaQ rnhA θ β τa holE dnaN dnaX γb δ δ’ χ ψ dnaX holA holB holC holD Polymerization (catalytic subunit). 3’ → 5’ editing exonuclease. Removes RNA primers? Endonuclease that cleaves RNA in RNA/DNA duplexes. Primer removal in eukaryotes. Present in core. Sliding clamp. Organizes complex; joins leading and lagging DNA PolIII complexes. Clamp loading. Clamp loading. Clamp loading. Clamp loading. TABLE 1.1 Clamp loading. a b Full-length translation product of dnaX gene Shorter product of dnaX gene produced by translational frameshifting (Chapter 2) Some Properties of DNA Polymerases 1. All DNA polymerases require a template and a PRIMER (3’-OH end). Primers can be DNA or RNA. 2. In addition to the ability to replicate DNA in the 5'→3' direction, the DNA polymerases have a 3'→5' exonuclease activity (for proof-reading). 3. Pol I & II also have a 5'→3' exonuclease activity for the degradation of primers or widening of gaps during DNA repair (Pol III may lack this activity). IMPORTANT NOTE: RNA polymerases do NOT require a primer for the initiation of RNA synthesis! Primases are RNA polymerases. OH Functions of the primer and template during DNA synthesis Fig. 1.7 OH C The Basics of DNA Replication 1. Replication starts at origins of replication (ori sites). 2. Once initiated, replication proceeds bidirectionally on both template strands by leading- and laggingstrand synthesis. 3. Leading-strand synthesis is continuous (5’ Æ 3’). 4. Lagging-strand is discontinuous and involves the synthesis and joining of many “Okazaki” fragments. All Okazaki fragments are synthesized in the same orientation (5’ Æ 3’). 5. Replication ends when replication complexes collide. However the regions in the genomes where collisions occur may be determined by replication-termination (ter ) sites. DNA Replication: Initiation 1. DNA replication starts at origins of replication (ori ) AT-rich 12-bp 3 AT-rich 13-mers 4 DnaA recognition sites Fig 1.16 The ori-C site of E. coli is slightly less than 260 bp long and includes 4 DnaA boxes, 3 AT-rich 13-mers, a 12-bp AT-rich strandseparation region, and 11 GATC/CTAG Dam methylation sites. Dam = deoxyadenosine methyl transferase What is a “site”? There are sites within sites! What is a “box”? DNA Replication: Initiation oriC 10 to 12 molecules of DnaA protein with ATP bind to the DnaA boxes of oriC and form a complex that winds DNA around itself and causes opening of the helix in the adjacent AT-rich regions. 3’ 5’ DnaC protein delivers and clamps DnaB helicase protein as a hexamer to each strand in the open DNA 3’ “bubble”. 5’ 3’ DnaG primase synthesizes primers initiating replication. 3’ 5’ 5’ 3’ 5’ Primosome SSB 5’ 3’ Helicase begins unwinding more DNA by sliding along each strand in the 5’ Æ 3’ direction. Strands are kept separate by binding of single strand binding protein (SSB). Bidirectional Replication 2. Replication starts at origins of replication and proceeds bidirectionally along both strands of the helix until one replication complex collides with another going in the opposite direction. θ (theta) DNA structure DNA Replication: Elongation Model for leading and lagging strand replication GTC – sites for the initiation of RNA primer synthesis: 4 sites shown DNA gyrase (a type II topoisomerase) Dna B helicase DNA Pol III DIMER Primer RNA DNA helicase unwinding DNA MORE 3’ 5’ DNA Replication: Elongation Model for leading and lagging strand replication Okazaki fragment ~1000 b DNA G primase makes new RNA primer Primer degraded and gap filled by DNA Pol I LINK Videos Organization and dynamics of the E. coli replisome helicase primase clamp Modified from Fig. 4 Johnson and O'Donnell (2005). Annu. Rev. Biochem. 74:283-315. Completion of Lagging Strand Synthesis A. DNA Pol III starts from a primer and replicates until it reaches the primer of the previous Okazaki fragment. B. DNA Pol III is released from lagging strand and “snaps back” to nearest unused primer to start new Okazaki fragment. C. DNA polymerase I degrades primer between Okazaki fragments in 5’ to 3’ direction and fills gap with DNA. D. DNA ligase seals last gap. DNA polymerase III DNA polymerase I DNA ligase ATP ADP + Pi A Summary of DNA Replication Termination of Replication 5. Termination of replication occurs when replication complexes collide. At least some prokaryotes have termination sites (ter ), which are inverted repeats of many short repeats of nucleotide sequences that bind proteins and progressively slow down the movement of replication forks (replication complexes). The ter sites are not essential: deletion of these sites from the Bacillus subtilis chromosome slows cell growth but does not block cell division. Eukaryotes and viruses may not have ter sites. Fig 1.18 Stalling of fL Tus (E. coli ), RTP (B. subtilis) are replicationtermination proteins that bind to ter sites Stalling of fR Replication of Linear Chromosomes: the end problem Why is there no primer to synthesize DNA at the ends of chromosomes? There is no template for the primase to make a primer! Why can't DNA polymerase replace the RNA primer? It can not synthesize DNA in the 3’ 5’ direction and it can not initiate replication in the 5’ 3’ direction unless it is primed! Replication of Linear Chromosomes Some solutions to the end problem 1. The linear chromosomes of some bacteriophages have singlestranded cohesive ends and are circularized after entry into the host cell. Example: The ~48,000-bp phage λ chromosome. cos sequence cos cos Replication of Linear Chromosomes The ends of the linear chromosomes and plasmids of some organisms are covalently-closed hairpin structures. TIR Borrelia burgdorferi TIR African swine fever virus Vaccinia virus L ~25-bp inverted repeat R Replication initiated from internal origins Replication of Linear Chromosomes with Hairpin Ends Replication produces a circular dimer “Telomere resolvase” recognition site Telomere resolvase cleaves and ligates appropriate ends to form hairpins Replication of Linear Chromosomes: Invertrons ITR 5’ TP (Terminal protein) tp pol 3’ Terminal Protein O OH Ser Thr or Tyr Replication of the ends of linear chromosomes of the invertron type is primed by a nucleotide linked to a protein (terminal protein = TP). The TP remains permanently attached to the 5’ termini. Examples: Streptomyces, B. subtilis Φ29 3’ Replication of Linear Chromosomes Some solutions to the end problem 4. Eukaryotes solve the endreplication dilemma by extending their 3’ end by means of of a reverse transcriptase that has its own RNA template: telomerase. Second-strand synthesis require primase and DNA polymerase Replication RNA Primer Some telomeres are 100 to 200 Kb long Replication Error Editing and Methyl-directed Repair Nick Translation A 5’Æ3’ exonuclease 3’ 5’ RNA 5’ 3’ B Fig 1.11 DNA polymerase I can remove the nucleotides of an RNA primer (or nicked DNA strand) and simultaneously fill the gap with a new DNA strand by virtue of its “nick-translation” activity. C 5’Æ3’ exonuclease Pol Exo Nick translation Mistakes in base pairing can lead to changes in the nucleotide sequence of DNA: mutations Fig 1.13 GMT mismatch During replication (unedited) dsDNA with One mismatched base pair Replication Wild type Mutant Proofreading and Editing A mismatch in pairing at the terminal base pair causes the DNA polymerase to pause: the last base in the new strand is removed by the 3’ Æ 5’ exonuclease activity of DnaQ, the ε subunit of DNA polymerase III, before replication continues. Proofreading Removal of mismatched base Error corrected as synthesis is resumed Proofreading and Editing DnaQ can remove only mismatched nucleotides that are located at the 3’ end of the newly synthesized strand. If the polymerase adds even one additional nucleotide, then DNA Q no longer can remove the mismatched base. This situation can result in a mutation, unless the damage is repaired by a different mechanism. What is a mutation? A mutation is a heritable change in the nucleotide sequence of the genome of an organism. As we will see later in this class, there are many lesions in DNA molecules, most of which can be repaired. Only a small fraction of DNA lesions actually result in mutations. If a mismatch escapes repair by replication editing, it still can be fixed by methyl-directed DNA mismatch repair. In E. coli, newly-synthesized DNA strands can be distinguished from “old” template strands by delayed methylation of A in 5’GATC-3’ sites by deoxyadenosine methyltransferase (DAM). CH3 5’ 3’ GATC CTAG CH3 Both strands methylated Pol III Pol III 3’ old 5’ new 3’ new 5’ old Hemimethylated DNA Post-replication base modifications are mechanisms involved in: 1. Methyl-directed DNA mismatch repair. 2. Protection of DNA from cleavage by cellular site-specific restriction endonucleases. 3. Gene expression in some prokaryotes and all eukaryotes. Methyl-directed mismatch repair Dam targets Dam = deoxyadenosine methyl transferase Cut? MutG cuts at nearest unmethylated GATC. UvrD helicase unwinds strands past mismatch. New strand is degraded in the 5’Æ3’ direction by RecJ or ExoVII if nick is on 5’ side, or in the 3’Æ5’ direction, or by ExoI or ExoX if the nick is on the 3’ side of the mismatch. C T Cut? Dam adds methyl to 6 position of A DNA pol III MutS, MutL and MutG endonuclease bind to mismatched bp. Chromosome Partitioning into Daughter Cells Replication and Partitioning SeqA at hemimethylated GATC sites? Fig 1.19 FtsZ ring The binding of SeqA to hemimethylated GA*TC/CTAG sequences protects against premature initiation of replication before the cell cycle is complete. SeqA Visualization of hemi-methylated DNA at oriC by binding of SeqA-YFP protein (detected by yellow fluorescence in UV light by confocal microscopy). SeqA probably is NOT directly involved in binding of oriC to the membrane Jellyfish have yellow and green fluorescent proteins. Fig 1.21 The polymerization of the FtsZ tubulin into a ring determines the site of cell division and is critical for the correct distribution of daughter chromosomes during cell division (cytokinesis). FtsZ Ring (FtsZ-GFP) (Landing pad for recruitment of other proteins to division site -GTPase, required for cytokinesis) Modulators of FtsZ ring, connecting the ring to the cytoplasmic membrane Coordination of chromosome distribution with cell division, ATPdependent DNA translocase, binds Topo IV (decatenase) and XerCD (dif, dimer resolvase) Peptidoglycan synthase Peptidoglycan hydrolase Escherichia coli, Bacillus subtilis and Caulobacter crescentus all divide by assembling an FtsZ tubulin ring, but each species uses different proteins and mechanisms to direct FtsZ to its proper location. NOTE the polar proteins, which oscillate from pole to pole in E. coli. Helical filaments formed inside the cell membrane by MreB, an actin-like protein, are involved in the oscillation of the Min proteins. MreB and ParM are Actin Homologs MreB is always encoded by a gene on the bacterial chromosome and controls a wide variety of cellular functions, including: 1. Cell shape (mutants form spherical rather than elongated cells). 2. Localization of polar proteins (proteins are mislocalized in mutants) 3. Chromosome segregation (chromosomes are not separated into daughter cells in mutants). MreB self assembles into filaments in vitro. In vivo, MreB forms bundles of filaments that are arranged in a helical structure inside the cell. Like the actin filaments of eukaryotic cells, MreB filaments appear to assemble and disassemble from opposite poles (ends). The ParM actin-like protein always is encoded by genes located on plasmids and is dedicated to the proper partitioning of plasmid DNAs during cell division. What have we learned from the partitioning of Plasmids? Many plasmids have partitioning systems that prevent curing by random loss. Single-copy plasmids such as P1, F, and R1 have partitioning systems that consist of the following components: 1. A cis-acting “centromere-like” binding site on the plasmid DNA, parC. 2. Two proteins, the actin-like ParM and a parC-binding protein, ParR. Multiple copies of ParR, or its equivalent in other plasmids, bind to parC, a centromere-like sequence found in single-copy plasmids, forming the equivalent of a “kinetochore”. The parC/ParR complex then promotes polymerization of ParM into filaments between the two plasmids, pushing them from the middle to the quarter positions of the dividing cell. Like actin, ParM has ATPase activity. R1 plasmid segregation F plasmid segregation SopA aster Aster oscillates between kinetochores ParM From Z. Gitai 2006. Curr. Biol. 16: R133-R136. What does plasmid segregation tell us about bacterial chromosome segregation? A lot, but not the whole story... 1. Bacteria have several genes that code for homologs of the Par proteins of plasmids, including MreB (many bacteria), called Soj in Bacillus subtilis. 2. The MreB proteins of bacteria are actin homologs. 3. In Bacillus subtilis, filaments of a ParM-like protein, Soj, are linked to a parC-like nucleotide sequence near ori through multiple copies of a ParR-like protein, Spo0J. 4. Bacterial partitioning proteins also show “oscillatory cycles” in vivo similar to the SopA asters of the F plasmid. 5. Electron cryotomographic examination of bacterial cells has revealed exquisite internal compartmentalization, including a cytoskeleton consisting of numerous bundles of filaments similar to those of the cytoskeleton of eukaryotic cells. Resolution of Chromosome Dimers and Concatemers dif Chromosome dimers are generated by homologous recombination during replication dif dif dif Dimer dif dif dif dif dif dif Chromosome dimers are resolved by XerCD resolvase through site-specific recombination at dif during cytokinesis. Question for Thought What happens if two circular DNA molecules, such as bacterial chromosomes or plasmids, are joined by two, three or more “crossovers” (recombination events) that have occurred at different sites? Can you develop a rule about the structures that are produced by multiple recombination events? Which of these structures would require XerCD site-specific recombination at dif for their resolution during cytokinesis? Concatemers of Chromosomes are Resolved by type II Topoisomerases (Topo IV in E. coli ) DNA Packaging in Nucleoids Electron micrograph of uranyl acetate/lead-citrate stained thin section of an E.coli cell showing electron-dense (clear) regions of condensed DNA Nucleoid Fig. 1.22 NOTE: The Planctomycetes are prokaryotes that have complex internal membrane structures. Members of the genus Gemmata have nucleoids that are enclosed by an envelope resembling nuclear membranes of eukaryotes. From J. A. Fuerst: Intracellular Compartmentation in Planctomycetes. Annu. Rev. Microbiol. 2005. 59:299–328 Organization of Prokaryotic Chromosomes Nucleoid Plasmid DNA DNA is tightly compressed into nucleoids and spreads when cells are gently lysed. Chromosome Compacting of DNA into a Repetitive Stable Structure by E. coli MukBEF Condensin Hinge Condensation by sequential binding of MukBEF Arms ABC Heads * Relaxation by extension and sequential release of MukBEF From: Case et al. Science 305: 222-227 (2004) * ABC = ATP-binding cassette Speculative Model of Condensed Prokaryotic Chromosome ~30 kb domains of supercoiled DNA protruding from between heads of MukBEF dimers Helically propagated MukBEF complexes From: Case et al. Science 305: 222-227 (2004) Electron micrographs of bacterial nucleoids showing supercoiled and partially-relaxed loops of DNA Partially relaxed Supercoiled Highly supercoiled Supercoiled DNA Nick Break one strand Seal Break one strand Relaxed covalently closed circular DNA Relaxed nicked circular DNA Rotate one end of broken strand around helix and seal Proteins Inside cells, DNA is highly supercoiled associated with proteins. MORE Supercoiled circular DNA Supercoiled domain A Supercoiling and relaxation of helical double-stranded DNA Fig. 1.24 Enzymes are necessary to increase or decrease DNA supercoiling: gyrases and topoisomerases Topo I Fig 1.25 Topo II and gyrase Such enzymes are essential for replication and DNA repair Gyrase (Topo II) Action DNA dimer 5’ PO4 ends linked to tyr122 of GyrA subunits. From Costenaro, L., Grossmann, J.G., Ebel, C. and Maxwell, A. Small-angle X-ray scattering reveals the solution structure of the full-length DNA gyrase a subunit., Structure 13: 287–296, 2005 Topo I Topo II and gyrase Summary of Supercoiling and Topoisomerases Positive and negative supercoiling can occur in the same DNA molecule due to the action of helicases, gyrases and topoisomerases. Double-stand “breaks” by Topo II to relax negative or positive supercoiling or gyrase (+ ATP) to add negative supercoiling Any helicase Topo I single-strand nicks relax + or − coiling SV40 replisome Coordination of Chromosome Replication with the Rate of Cell Division Cells grow and divide at different rates in different media. For example, the doubling times for E. coli are: ~ 140 min in succinate minimal medium. ~ 70 min in glucose minimal medium. ~ 30 min in Luria broth. The rate of DNA synthesis in E. coli is the same in all media: approximately 230,000 nucleotides are polymerized by the 4 DNA pol III complexes involved in bidirectional replication. The E. coli chromosome is a 4.63 Mbp circle. It takes ~40 min to complete one round of DNA replication. Therefore, rapidly dividing cells would lose chromosomes, and slowly dividing cells would accumulate DNA, unless chromosome replication is synchronized in some way with the rate of cell division. Coordination is accomplished by controlling the initiation of rounds of replication. Coordination of chromosome replication with generation time I = 70 min I = 30 min Fig 1.20 Antibiotics that Block DNA Replication (a quinolone) Ciprofloxacin Chemically synthesized fluoroquinolone GyrA of DNA gyrase (also other Topo II) WARNING! The indiscriminate use of Cipro already has resulted in extensive selection of bacterial pathogens that are resistant to fluoroquinolone antibiotics, even though it was thought that it was “virtually impossible” for microbes to acquire the multiple mutations that were deemed necessary to develop this trait. Fig 1.5 Synthesis of deoxyribonucleotides from ribonucleotides Abbreviations: THF, tetrahydrofolate DHF, dihydrofolate Inhibitors of DNA Synthesis Ciprofloxacin 4-quinolone Mitomycin C Nalidixic acid DNA remains linked to tyr122 of GyrA subunits; gaps not closed. Novobiocin (a coumarin) Crosslings DNA strands Competitive inhibitor of ATPase activity of GyrB