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DNA replication

by: Hannah Welsh
Hannah Welsh


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This is all there is to DNA replication in bacteria and eukaryotes as discussed in the textbook
Dr. Cheryl Ingram-Smith
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This 9 page Class Notes was uploaded by Hannah Welsh on Friday March 11, 2016. The Class Notes belongs to 3010 at 1 MDSS-SGSLM-Langley AFB Advanced Education in General Dentistry 12 Months taught by Dr. Cheryl Ingram-Smith in Spring 2016. Since its upload, it has received 15 views.


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Date Created: 03/11/16
DNA Replication >DNA Replication is Semi-Conservative: one strand is a template for making a new DNA strand, and the new strand is a one-old, one-new strand hybrid Meselson and Stahl, 1957: Grew E. coli in heavy nitrogen The E. coli that had grown up in the 15N medium were transferred to 14N medium, where they were grown until the population had just doubled. The DNA isolated from the first 14N generation had one band in 15-14N hybrid area. With another cycle of replication in the light 14N medium, there was a band at the 15-14N area, as well as the 14N light nitrogen isotope area. >Replication Begins at an Origin and Usually Proceeds Bidirectionally One or both ends of the circular bacterial chromosome in Cairns’s experiment were dynamic points called replication forks, in which parent DNA is being unwound and the separated strands, quickly replicated—he found that both strands were replicated at the same time, and that both ends of loop have active replication forks—replication proceeds in both directions -replication starts at one unique point, an origin. For circular DNA molecules: two replication forks meet at a place on the circle opposite the origin >DNA goes from 5’ to 3’ and is semi-discontinuous -DNA synthesis must begin with a free 3’ OH as the point for DNA elongation to occur -DNA is antiparallel, so if a strand is a template, it is read from the 3’ end to the 5’ end, in order that synthesis can proceed in 5’ to 3’ Okazaki and Co., 1960s: one of the new DNA strand being synthesized is made in bite-size fragments—the lagging strand—while the other is synthesized continuously, the leading strand, because its synthesis is in the same direction as the movement of the replication fork >DNA is degraded by Nucleases Nucleases, or Dnases, are specific for DNA and not RNA, and cells contain several different types of general “nucleases”—there are exonucleases that degrade nucleic acids from a single end of DNA, many removing nucleotides in only one direction (5’/3’ or 3’/5’) from a DNA strand Endonucleases can start degrading at internal sites in a nucleic acid strand, molecule, and leave a bunch of fragments -some nucleases are specific to single-stranded DNA only, some endonucleases only cut at specific nucleotide sequences >DNA is synthesized by DNA Polymerases Kornberg and Co., 1955: purified DNA polymerase I from E. coli (encoded by polA gene). *Fundamental reaction of DNA polymerases—a phosphoryl group transfer Nucleophile: 3’ OH group of the nucleotide at the 3’ end of the growing DNA strand. Nucleophilic attack occurs at the alpha-phosphorus of the incoming deoxynucleoside 5’-triphosphate Catalysis: needs two Mg2+ ions at the active site: 1—deprotonates the 3’ OH group, making it a better nucleophile 2—Binds to the incoming dNTP and helps get the pyrophosphate out of the way -minimal free energy transfer; you give up a less stable phosphate anhydride to make a more stable phosphodiester bond—noncovalent base-stacking and base- pairing interactions will help stabilize lengthening DNA relative to free nucleotides (+hydrolysis of the pyrophosphate by pyrophosphatase helps form products as well) All DNA polymerases must have a template, all DNA polymerases require a primer -a primer: a strand piece with a free 3’ OH group to which you can add free nucleotides (the free 3’ end is the “primer terminus”). Many primers are oligonucleotides of RNA rather than DNA; special enzymes can make primers for the DNA polymerase to work off of DNA polymerase active sites have two parts 1. insertion site, where you put the incoming nucleotide 2. post-insertion site, where the DNA polymerase slides to next after the phosphodiester bond is formed with the incoming nucleotide, and where the new base pair is positioned DNA polymerase adds nucleotides to the growing DNA strand, either continuing down the template and adding nucleotides, or dissociating Processivity: the average number of nucleotides that can be added before a polymerase dissociates REPLICATION IS VERY ACCURATE The DNA POLYMERASE discriminates not just between correct/incorrect nucleotides based on hydrogen-bond pairing between the base complements, but also on the “geometry” of the standard A--T and G---C pairs Active site of DNA Polymerase I will only accommodate base pairs with their correct geometry -incorrect nucleotides may be able to hydrogen bond with a base in the template, but it cannot then fit in the active site, and so that means you can reject the wrong base before the phosphodiester bond is formed Accuracy of polymerization isn’t the whole story on DNA replication fidelity, however; it can still be fooled by bases in an unusual tautomeric form that can hydrogen-bond with an incorrect partner 3’-5’ exonuclease activity can double-check nucleotides that have already been added. It lets an enzyme remove a newly-added nucleotide, and it is highly specific for mismatch repair If polymerase added the wrong nucleotide: the translocation of the polymerase to the next position is inhibited, and the “kinetic pause” lets you correct the mistake. 3’-5’ exonuclease activity removes the mispaired nucleotide, and the DNA polymerase keeps on going—this is known as proofreading, and is not merely the “reverse of polymerization”—for the reason that no pyrophosphate is involved E. COLI HAS AT LEAST FIVE DNA POLYMERASES—BUT MOST ACTIVITY IS FROM DNA POLYMERASE I -But since DNA Polymerase I’s rate of adding nucleotides is too slow, and it has low processivity (relatively), it cannot be the suitable thing for replicating the large E. coli chromosome 1970s: DNA Pol II and DNA Poly III discovered; DNA polymerase III was found to be the principal replication enzyme in E. coli DNA Polymerase I provides cleanup functions during replication/recombination/repair—which is enhanced by 5’-3’ exonuclease activity -which is located in a structural domain that can be removed from the rest of the enzyme, leaving behind the “large fragment” or “Klenow fragment” which can still do polymerization and proofreading. 5’-3’ exonuclease activity of intact DNA Poly I can replaced a segment of DNA or RNA paired to the template strand— nick translation -DNA Polymerase III is much more complicated than DNA Polymerase I alpha and epsilon subunits: polymerization and proofreading, respectively alpha and epsilon and theta: core polymerase—which can polymerize DNA with limited processivity -Two core polymerases can be linked by the clamp-loading, or gamma complex, which has five subunits of four different types (tau2gammadeltadeltaprime) -core polymerases are linked through tau subunits -the clamp-loading complex has two additional subunits, chi and psi, bound to the clamp-loading complex ALL 13 PROTEIN SUBUNITS GIVES YOU DNA POLYMERASE III -DNA Polymerase III is absolutely able to polymerize DNA, but with much lower processivity than would be needed to replicate the chromosome—the solution? The addition of BETA subunits, four of which complete the DNA Poly III holoenzyme -Beta subunits: encircle DNA and clamp onto it. Each dimer goes with a core subassembly of polymerase and slides along the DNA as replication goes on -Beta sliding clamp prevents dissociation of DNA polymerase III from DNA, increasing processivity DNA REPLICATION NEEDS SEVERAL ENZYMES AND PROTEIN FACTORS Replication in E. coli is misleading, because you don’t just need one little DNA polymerase, but a whole host of 20 or more different enzymes and proteins doing a certain task—this comprises the DNA replicase system or replisome One main hurdle is getting the two parent strands apart so you can use one as a template—which is accomplished by helicases, enzymes that slide along DNA and separate DNA strands, using energy from ATP. -Strand separation creates “topological stress” in the DNA helix, which is relieved by topoisomerases. -Then-separated strands are stabilized by DNA-binding proteins As we know, DNA polymerases can’t synthesize DA unless the template’s got some primers on it first. Segments of RNA can be synthesized on the DNA template by enzymes called primases. The RNA primers will be removed and replaced by DNA at sometime in the replication process—in E. coli, this is the job of DNA Polymerase I -After RNA primers are removed and the gap is filled in with DNA, you still have a nick in the DNA backbone—a broken phosphodiester bond—which is then sealed by an enzyme called DNA ligase REPLICATION OF THE CHROMOSOME IN E. COLI -INITIATION-ELONGATION-TERMINATION Initiation: E. coli replication origin—oriC. The replication origin has several DNA sequence elements that are found to be highly conserved among bacterial replication origins -five repeats of a 9 base pair sequence—R sites—serve as binding sites for the key initiator protein DnaA -region rich in A--T called the DNA unwinding element. There are three additional DnaA-binding sites (I sites) and binding sites for the proteins IHF (integration host factor) and FIS (factor for inversion stimulation). These two proteins were discovered to be necessary for certain recombination reactions. -another DNA-binding protein, HU, also participates, but does not have its own binding site ENZYMES—open the DNA helix at the origin, and make a “prepriming complex” so that the next reactions can happen Crucial component in the initiation of replication is the DnaA protein, a member of the AAA+ ATPase protein family. Many AAA+ ATPases, including DnaA, form oligomers and hydrolyze ATP—slowly. \ *The slow hydrolysis of ATP acts as a switch mediating interconversion of the protein between two states—the ATP-bound form of DnaA being active, and the ADP-bound form, inactive Eight DnaA protein molecules in the active, ATP-bound state, assemble to make a helical complex encompassing the R and I sites in the oriC replication origins -DnaA has more of an affinity for R than I sites, and binds R in the inactive or active form equally well. The I sites only bind the ATP-bound DnaA, allowing discrimination between active and inactive DnaA forms *Tight wrapping of DNA around this protein complex gives a positive supercoil, and the strain triggers denaturation in the AT rich DNA unwinding element region (complex at the replication origin also includes several DNA-binding proteins—HU, IHF, and FIS—that help DNA bind better) DnaC, another AAA+ ATPase family member, loads the DnaB protein onto the separated DNA strands, within the denatured region. A hexamer of DnaC, with an ATP bound to each subunit, forms a tight complex with the hexameric, ring-shaped DnaB helicase DnaB and DnaC interactions opens up the DnaB ring (aided by further interaction between DnaB and DnaA). Two of the ring-shaped DnaB hexamers will load on the DNA unwinding element ATP bound to DnaC will hydrolyze, releasing the DnaC and leaving DnaB on the DNA Loading DnaB helicase is the KEY STEP in replication initiation. DnaB will migrates on ssDNA in the 5’-3’ direction, unwinding DNA as it goes. The DnaB helicases loaded on the two DNA strands will travel in opposite directions, making two potential replication forks *all other proteins at the replication fork are somehow, indirectly or directly, bound to DnaB Many molecules of single-stranded DNA-binding protein (SSB) will bind to and stabilize single DNA strands and keep them separated DNA gyrase (aka, DNA topoisomerase II) will relieve the topological stress made up ahead of the replication fork as the DNA unwinds -Initiation is the only known regulated phase of DNA replication, and is carefully controlled so that it only occurs once per cell cycle -Once DNA polymerase III is loaded onto DNA, along with the beta subunits, the protein Hda binds to the beta subunits and interacts with DnaA to stimulate hydrolysis of the ATP bound to the betas -Had is another AAA+ ATPase member, closely related to DnaA -ATP hydrolysis in this case makes the DnaA complex dissociated at the origin. Slow release of ADP by DnaA and rebinding of ATP cycles the protein between inactive—bound ADP—and active—bound ATP—forms on a time scale of 20 minutes to 40 minutes What affects timing of replication? DNA methylation and interactions with the bacterial plasma membrane *oriC replication origin is methylated by DNA methylase that methylates a certain adenine residue—oriC is methylated by Dam methylase (DNA adenine methylation) -Immediately after replication, DNA is hemi-methylated: the parent strands have methylated oriC sequences, but the new strands do not—an unknown mechanism sequestered the hemi-mehtylated sequences in the plasma membrane, and by the binding of the protein seqA *After a time, oriC is released from the plasma membrane, seqA dissociates, and you’d have to fully methylate the DNA with Dam methylase before it can bind DnaA again and initiate a new round of replication Elongation: includes leading strand synthesis and lagging strand synthesis. Parent DNA gets unwound by DNA helicases, and the stress is relieved by topoisomerases. Each separated strand is stabilized by SSB. Leading strand: begins with synthesis by primase aka DnaG protein (primase) that makes a short RNA primer at the replication origin. DnaG-DnaB interaction helps carry out this reaction, synthesizing the primer in the direction opposite to that which the DnaB helicase is moving. *Effectively, DnaB helicase moves along the strand that will be the lagging strand, while the first primer laid down in the first DnaG-DnaB interaction primes leading strand synthesis in the opposing direction *deoxyribonucleotides are added to the primer by a DNA Polymerase III complex linked to the DnaB helicase tethered to the opposite DNA strand Lagging strand: Firstly, an RNA primer is synthesized by primase (DnaG) and, as in leading strand synthesis, DNA polymerase III binds to the RNA primer and adds deoxyribonucleotides -This gets complicated when you have to coordinate leading and lagging strand synthesis; both strands are made by ONE, asymmetric DNA polymerase III dimer, and so you have to physical loop the DNA of the lagging strand over to get the polymerization to happen at one time in the right directions Okazaki fragments: DnaB helicase and DnaG primase together make a unit within the replication complex called the primosome. DNA polymerase III uses one set of its core subunits to do continuous synthesis on the leading strand, while the other set of core subunits cycles form one Okazaki fragment to the next on the lagging strand -DnaB helicase, as it is in front of the polymerase, unwinds the DNA at the replication fork, and it travels down the lagging strand template in the 5’-3’ direction. AS it does this, DnaG primase occasionally comes in for a landing and synthesizes a short RNA primer -A new beta sliding clamp is then positioned at the primer by the clamp-loading complex of DNA polymerase III. When an Okazaki fragment is done, replication stops, and the core subunits of DNA polymerase III dissociate from their beta sliding clamp and from the Okazaki fragment—they then associate with a new clamp and initiate synthesis of another Okazaki fragment Clamp-loading complex of DNA Poly III: has parts of the two TAU subunits, along with the gamma, delta, delta prime subunits—the clamp-loading complex belongs to the AAA+ ATPase family -The clamp-loading complex binds to ATP and to the new beta sliding clamp. Binding strains the clamp, break open the ring of the clamp at one subunit face, so that you can slip the newly-primed lagging strand into the ring through the break. The clamp-loader will hydrolyze ATP, releasing the beta sliding clamp/letting it close around the DNA -The replisome (entire complex that coordiantes DNA synthesis at a replication fork) promotes fast DNA synthesis >Once you’ve finished an Okazaki fragment, the leftover RNA primer is removed and replaced with DNA by DNA polymerase I; the remaining nick is sealed with DNA ligase. DNA ligase: catalyzes the formation of a phosphodiester bond between a 3’ hydroxyl at one end and a 5’ phosphate at the other end of the DNA strand—but first, the phosphate must be activated by adenylation (using ATP, in eukaryotes and viruses; bacteria use NAD+) Termination: When the two replication forks of the circular E. coli chromosome meet, they meet at terminus region containing many copies of a 20 base-pair sequence called Ter. -Ter sequences are arranged to create a “trap” that the replication fork can enter, but cannot leave. Ter sequences function as the binding sites for the protein Tus (terminus utilization substance). The Tus-Ter complex can arrest a replication fork from only ONE DIRECTION —this is why only one Tus-Ter complex functions per one replication, because it’ll stop at the complex first encountered by the replication fork Opposing replication forks have to stop when they collide, so why are Ter sequences necessary? They may prevent overreplication at one fork if the other fork is slowed by DNA damage or another obstacle The first replication fork to get to a Tus-Ter complex will halt, and the other fork halts when it catches up the first, arrested fork. The final, few base pairs of DNA between the protein complexes are then replicated (by an unknown mechanism), which makes two topologically linked chromosomes—called catenanes -Separation of catenated chromosomes is done by topoisomerase IV, which allows the chromosomes to be segregated into daughter cells when the cell divides REPLICATION IN EUKARYOTIC CELLS IS SIMILAR BUT MORE COMPLEX Origins of replication are more defined in lower eukaryotes, but have less defined structures in higher eukaryotes. In vertebrates, a variety of AT rich sequences may be used for replication initiation, and the sites can vary from one cell division to the next *Yeast: have defined replication origins called “autonomously replicating sequences,” ARS, or replicators >Regulation ensures that all cellular DNA is only replicated once per cycle Licensing: formation of the pre-replicative complex, making the cell ready for replication (fast-growing cells show pre-RCs at the end of M phase, slow-growing cells don’t show them until the end at G1) Initiation key in eukaryotes: as in bacteria, the key event is the loading of the helicase, which, in eukaryotes, is a heterohexameric complex of mini- chromosome maintenance proteins—the ring-shaped MCM2-7 helicase I (analogous to the bacteria’s DnaB helicase) is loaded onto DNA by a six-protein complex called the origin recognition complex ORC has five AAA+ ATPase domains along its subunits—it’s basically like the bacterial DnaA; tow other proteins are also required to load the helicase complex >Commitment to replication requires synthesis of active S-phase cyclin- CDK complexes *these proteins bind to and phosphorylate several proteins in the pre- replicative complexes; other processes prevent more pre-RCs from being made, beyond the ones already licensed >Eukaryotic chromosomes have multiple origins of replication DNA polymerase alpha: responsible for the replication of nuclear chromosomes. It is a multisubunit enzyme: one subunit has primase activity, and the largest subunit has polymerization activity—but this polymerase does not have 3’-5’ exonuclease activity, and so it is unsuitable for high-fidelity DNA replication—we think that DNA polymerase alpha is only used to synthesize short RNA primers on the lagging strand for Okazaki fragments -These primers are then extended by DNA polymerase delta, aided by PCNA, which acts like the beta sliding clamp in prokaryotes—it forms a circular clamp that greatly increases the enzyme’s processivity. DNA polymerase delta does have 3’-5’ exonuclease activity Termination: involves synthesis of telomere structures on the ends of chromosomes DNA REPLICATION SUMMARY -Replication of DNA is high-fidelity and occurs once at a designated time in the cell cycle -DNA replication is semi-conservative: each old strand is a template for a new daughter strand (Meselson-Stahl experiment) -three phases to DNA replication: initiation, elongation, termination -DNA replication is bidirectional (starts from one origin in bacteria) -DNA synthesis in DNA replication runs in the 5’-3’ direction, done by DNA polymerases -At the replication fork, leading strand synthesis is continuous, while lagging strand synthesis is discontinuous and done in Okazaki fragments (which are then ligated) -DNA replication fidelity is due to: 1. base selection by the polymerase 2. 3’-5’ exonuclease activity by most DNA 3. specific repair for mismatches made during replication -Most cells have several DNA polymerases; E. coli use DNA Pol III primarily, and DNA Pol I is used for special functions in replication, recombination, and repair -Separate initiation, elongation, and termination phases of DNA replication involve a bunch of enzymes and protein factors, many in the AAA+ ATPase family -major replicative DNA polymerase in eukaryotes: DNA polymerase delta (DNA Pol alpha acts like a primase, and DNA Pol epsilon repairs DNA)


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