DNA ReplicationLec1415 - DNA Replication Structural...

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Unformatted text preview: DNA Replication Structural Overview of DNA Replication Existing DNA strands act as templates for the synthesis of new strands. DNA replication relies on the complementarity of DNA strands. The process can be summarized as follows (see fig. 11.1): The two DNA strands come apart (parental strands). Each serves as a template strand for the synthesis of new strands (daughter strands). Experiment 11A – Three different models were proposed that described the net result of DNA replication. s emi: after replication one strand is old, one is new, one strand is old one is new dispersive: each is made of one old and one new In the late 1950s, three different mechanisms were proposed for the replication of DNA (see fig. 11.2): Conservative model: both parental strands stay together after DNA replication. Semiconservative model: the double-stranded DNA contains one parental and one daughter strand following replication. nitrogen 15 has an extra nuetron compared t o nitrogen 14. Dispersive model: parental and daughter DNA is interspersed in both strands following replication. In 1958, Matthew Meselson and Franklin Stahl devised a method to investigate these models; they found a way to experimentally distinguish between daughter and parental strands. The hypothesis. Based on Watson’s and Crick’s ideas, the hypothesis was that DNA replication is semiconservative (see fig. 11.2b). Testing the hypothesis (see fig. 11.3). essentially all of the dna has heavy nitrogen, because all of the n t hat has been available f or generation after generation has been n-15 add 14 so that all will be 14 instead of 15 The starting material is a strain of E. coli that has been grown for many generations in the presence of 15N; therefore, all of the nitrogen in the DNA is labeled with 15N. Add an excess of 14N-containing compounds to the bacterial cells so that all of the newly made DNA will contain 14N. Incubate the cells for various lengths of time. 1 add cesium chloride goes to where density of molecule matches density of gradient heavy dna is gonna go to part. place of gradient--> just s eparating dna dna is all heavy --> put bacteria into medium with only light n. now all new stuff has light. this is only after one generation. Lyse the cells by adding lysozyme and detergent. Load a sample of the lysate onto a CsCl gradient. Centrifuge the gradients until the DNA molecules reach their equilibrium densities. Observe DNA within each gradient under UV light. after one gen. all dna is half heavy. consistent with both The data (see fig. 11.3). s emi cons. and dispersive models. Interpreting the data. after two generations. two t ypes of dna, light and half heavy. consistent with only s emi-conservative model. After one generation, the DNA is “half-heavy”; this is consistent with both semi-conservative and dispersive models. After two generations, the DNA is of two types: “light” and “half-heavy”; this is consistent with only the semi-conservative model. Bacterial DNA Replication Bacterial chromosomes contain a single origin of replication (see fig. 11.4). DNA synthesis begins at a site termed the origin of replication; each bacterial chromosome has only one. Synthesis of DNA proceeds bidirectionally around the bacterial chromosome. The replication forks eventually meet at the opposite side of the bacterial chromosome; this ends replication. Replication is initiated by the binding of DnaA protein to the origin of replication (see figs. 11.5 and 11.6). GATC - regulates when dna replication takes place one strand of old and one strand of new dna The origin of replication in E. coli is termed oriC – origin of chromosomal replication. Three types of DNA sequences in oriC are functionally significant: AT-rich region. DnaA boxes. GATC methylation sites. 2 helicases move antiparallel to one another synthesizes dna bidirectionally replication fork in c lockwise and in c ounterclockwise direction dna helicase is breaking the h bonds between single strand proteins bind to this and hold it open heicase causes + s upercoiling ahead of it, s o we need to releive + s upercoiling. gyrase/ topoisomerase II are s ame thing DNA replication is initiated by the binding of DnaA proteins to the DnaA box sequences and to each other; this binding stimulates the cooperative binding of an additional 20 to 40 DnaA proteins to form a large complex. Other proteins that cause the DNA to bend also bind; this causes the region to wrap around the DnaA proteins and separate the AT-rich region. The DnaA proteins, assisted by the DnaC protein, recruit DNA helicase enzymes (DnaB protein) to this site. When a DNA helicase encounters a double-stranded region, it breaks the hydrogen bonds between the two strands, thereby generating two single strands. Two DNA helicases begin strand separation within the oriC region and continue to separate the DNA strands beyond the origin. The DNA helicases separate the DNA in both directions, creating two replication forks outward from oriC in opposite directions; this initiates replication of the bacterial chromosome in both directions, an event termed bidirectional replication. - supercoiling is way able t it is compacted Several proteins are required for DNA replication at the replication fork (see fig. 11.7 and 11.1). Looking at one replication fork: dna polymerase cnanot s tart synthesizing dna. c an only add nucleotides on to already exisiting nucleotides dna primase creates a dna primer for dna. involved in synthesis of Rna primers synthesis is 5'->3' for dna and rna and that is c ontinuous lagging strand has mutliple primers in direction away from replication fork to replicate in pieces later primer will be replaced with dna DNA helicase separates the two DNA strands by breaking the hydrogen bonds between them; this generates positive supercoiling ahead of each replication fork. Topoisomerase (type II), also called DNA gyrase, travels ahead of the helicase and alleviates positive supercoiling. Single-strand binding proteins bind to the separated DNA strands to keep them apart. Replication REALLY begins here Short (10 to 12 nucleotides) RNA primers are synthesized by DNA primase; these short RNA strands start, or prime, DNA synthesis. The leading strand has a single primer, the lagging strand needs multiple primers; they are later removed and replaced with DNA. DNA polymerase III is responsible for synthesizing the DNA of the leading and lagging strands. DNA polymerase I excises the RNA primers and fills in with DNA. DNA ligase covalently links the Okazaki fragments together. 3 Details of DNA synthesis. dna pol I is a very s imple molecule c ompared to pol III the polymerase is moving along the s trand, not the reverse only in the 5' to 3' direction dna pol cant initiate it is rna primase (rna primers) DNA polymerase III is responsible for most of the DNA replication; it is a large enzyme consisting of 10 different subunits that play various roles in the DNA replication process (see Table 11.2). By comparison, DNA polymerase I is composed of a single subunit. Bacterial DNA polymerases may vary in their subunit composition (there are several others that will not be discussed at this time); however, they have the same type of catalytic subunit (see fig. 11.8). Structure resembles a human hand. Template DNA threads through the palm. build 5' phosphate to 3' hydroxyl of the deoxyribose of the nucleotide in front of it original template is 3' ->5'' leading 5' --> 3' in t erms of building lagging makes it 5' --> 3' in terms of building; as well in the backwards direction; discontinuously Thumb and fingers wrap around the DNA. Incoming dNTPs enter the catalytic site, bind to the template strand according to the AT/GC rule, and then are covalently attached to the 3’ end of the growing strand. DNA polymerases cannot initiate DNA synthesis – this problem is overcome by the RNA primers synthesized by primase; DNA polymerases can attach nucleotides only in the 5’ to 3’ direction – this problem is overcome by synthesizing the 3’ to 5’ strands in small fragments (see fig. 11.9). The two new daughter strands are synthesized in different ways (see fig. 11.10). Leading strand. One RNA primer is made at the origin. DNA polymerase III attaches nucleotides continuously in a 5’ to 3’ direction as it slides toward the opening of the replication fork. Lagging strand. Synthesis is also in the 5’ to 3’ direction; however it occurs discontinuously away from the replication fork. Many RNA primers are required; DNA polymerase III uses the RNA primers to synthesize small DNA fragments (1000 to 2000 nucleotides each) that are termed Okazaki fragments after their discoverer. 4 DNA polymerase I removes the RNA primers and fills the resulting gap with DNA; it uses its 5’ to 3’ exonuclease activity to digest the RNA and its 5’ to 3’ polymerase activity to replace it with DNA. After the gap is filled a covalent bond is still missing; DNA ligase catalyzes a phosphodiester bond thereby connecting the DNA fragments. DNA Polymerase III is a processive enzyme (see fig. 11.11). DNA polymerases catalyzes a phosphodiester bond between the innermost phosphate group of the incoming deoxynucleoside triphosphate and the 3’-OH of the sugar of the previous deoxynucleotide. In the process, the last two phosphates of the incoming nucleotide are released in the form of pyrophosphate (PPi). DNA polymerase III is a processive enzyme because it remains attached to the template as it is synthesizing the daughter strand. This processive feature is due to several different subunits in the DNA polymerase III holoenzyme: A complex of several subunits functions as a clamp loader that allows the DNA polymerase holoenzyme to initially clamp onto the DNA. The ! subunit, also known as the clamp protein, promotes the association of the holoenzyme with the DNA as it slides along the template strand. B s ubunits are used here, not to be c onfused with DNA pol B of Eukaryotic Replication that is a repair polymerase The effect of processivity is quite remarkable: In the absence of the ! subunit, DNA polymerase III falls off the DNA template after a few dozen nucleotides have been polymerized; its rate is ~ 20 nucleotides per second. In the presence of the ! subunit, DNA polymerase III stays on the DNA template long enough to polymerize up to 500,000 nucleotides; its rate is ~ 750 nucleotides per second. Replication is terminated when the replication forks meet at the termination sequences (see figs. 11.12 and 11.13). Opposite to oriC is a pair of termination sequences called ter sequences designated T1 and T2; T1 stops counterclockwise forks, T2 stops clockwise forks. The protein tus (termination utilization substance) binds to these sequences; it can then stop the movement of the replication forks. 5 t hey don't cross paths. t hey meet and everything is ligated t ogether DNA replication ends when oppositely advancing forks meet. Finally DNA ligase covalently links all four DNA strands. DNA replication often results in two intertwined circular molecules termed catenanes; these are separated by the action of topoisomerases. replisome is fourCertain c omponents enzymes of DNA replication bind to each other to form a complex (see fig. 11.14). DNA helicase and primase are physically bound to each other to form a complex called the primosome; this complex leads the way at the replication fork. two The primosome (DNA helicase + primase) is physically associated with the DNA polymerase holoenzyme forming the replisome. III Two DNA polymerase III proteins act in concert to replicate both the leading and lagging strands; the two proteins form a dimeric DNA polymerase that moves as a unit toward the replication fork. Because DNA polymerases can only synthesize DNA in the 5’ to 3’ direction, synthesis of the leading strand is continuous and that of the lagging strand is discontinuous. Lagging strand synthesis is summarized as follows: The lagging strand is looped; this allows the attached DNA polymerase to synthesize the Okazaki fragments in the normal 5’ to 3’ direction. Upon completion of an Okazaki fragment, the enzyme releases the lagging template strand and another loop is then formed. This processed is repeated over and over again. The fidelity of DNA replication is ensured by proofreading mechanisms (see fig. 11.15). DNA replication exhibits a high degree of fidelity; therefore, mistakes during the process are extremely rare (DNA polymerase III makes only one mistake per 100 million bases made). There are several reasons why fidelity is high: 6 Instability of mismatched pairs. Complementary base pairs have much higher stability than mismatched pairs; this feature only accounts for part of the fidelity and has an error rate of 1 per 1,000 nucleotides. Configuration of the DNA polymerase active site. DNA polymerase is unlikely to catalyze bond formation between mismatched pairs; this induced-fit phenomenon decreases the error rate to a range of 1 in 100,000 to 1 million. Proofreading function of DNA polymerase. DNA polymerases can identify a mismatched nucleotide and remove it from the daughter strand. t o remove an incorrect nucleotide uses a different doesn't start until dNAa boxes are coverd by dnaa protein exonucelase activity than to remove an RNA primer The enzyme uses its 3’ to 5’ exonuclease activity to remove the incorrect nucleotide and then changes direction and resumes DNA synthesis in the 5’ to 3’ direction. Bacterial DNA replication is coordinated with cell division. Bacterial cells can divide into two daughter cells at an amazing rate (E. coli can divide every 20 to 30 minutes); therefore, it is critical that DNA replication take place only when a cell is about to divide. Bacterial cells regulate the DNA replication process by controlling the initiation of replication at oriC; E. coli does this via two different mechanisms: The amount of DnaA protein provides one way to regulate DNA replication (see fig. 11.16). To begin replication, enough DnaA protein must be present to bind to all of the DnaA boxes; immediately following DNA replication, the number of DnaA boxes is double and there is not enough DnaA protein available to initiate a second round of replication. Another way to regulate DNA replication involves the GATC methylation sites within oriC (see fig. 11.17). DNA adenine methytransferase recognizes 5’–GATC–3’ sequences, binds to them, and methylates the adenine bases. two adenines in the s ite 7 c ontrolled by amount of dna protein and methylation at gatc sites Prior to DNA replication, these sites are methylated in both strands, and this full methylation of 5’–GATC–3’ sites facilitates the initiation of DNA replication at the origin. Following DNA replication, the newly made strands are not methylated, and initiation of DNA replication at the origin does not readily occur until after the 5’–GATC–3’ sites become fully methylated. radioactive nucleotides s tay in supernatant proteins/template dna radioactive nucleotides proteins-no template dna Experiment 11B – DNA replication can be studied in vitro. The in vitro study of DNA replication was pioneered by Arthur Kornberg in the 1950s; he received a Nobel Prize for his efforts in 1959. Kornberg hypothesized that deoxynucleoside triphosphates are the precursors of DNA synthesis. He also knew that these nucleotides are soluble in an acidic solution while long DNA strands are not. In this experiment, Kornberg mixed the following: An extract of proteins from E. coli. Template DNA. Radiolabeled nucleotides. These were incubated for sufficient time to allow the synthesis of new DNA strands. Addition of acid will precipitate these DNA strands. Centrifugation will separate them from the radioactive nucleotides. The hypothesis. DNA synthesis can occur in vitro if all the necessary components are present. Testing the hypothesis (see fig. 11.18). Mix together the extract of E. coli proteins, template DNA that is not radiolabeled, and 32P-labeled deoxyribonucleotide triphosphates; set up a control with no template DNA. Incubate the mixture for 30 minutes at 37ºC. Add perchloric acid to precipitate the DNA. 8 had template, radioactive, eznymes (proteins) Centrifuge the tube (unincorporated 32P-labeled deoxyribonucleotide triphosphates will remain in the supernatant). Collect the pellet, which contains precipitated DNA and proteins. Count the radiation in the pellet using a scintillation counter. The data (see fig. 11.18). 3,300 picomoles of radiation in the pellet of the complete system. No radiation in the control. Interpreting the data. E. coli proteins + nonlabeled template DNA + radiolabled nucleotides ! radiolabeled product. E. coli proteins + radiolabled nucleotides ! no radiolabeled product. Taken together, these results indicate that this technique can be used to measure the synthesis of DNA in vitro. we're bascially The s tudying what's different from a normal c ell, there must have been a point mutation in the gene or s omething like that isolation of mutants has been instrumental to our understanding of DNA replication. The isolation of mutants has been crucial in elucidating DNA replication; for example, mutants played key roles in the discovery of DNA polymerase III and various other enzymes involved in DNA synthesis. DNA replication is vital for cell division; thus, most mutations that block DNA synthesis are lethal; for this reason, researchers must screen for conditional mutants. A type of conditional mutant is a temperature-sensitive (ts) mutant; in the case of a vital gene, a ts mutant can survive at the permissive temperature, but it will fail to grow at the nonpermissive one. A general strategy for the isolation of ts mutants (see fig. 11.19): Expose bacterial cells to a mutagen and then plate on growth media; incubate at the permissive temperature. Plate cells from each colony onto replicate culture plates; incubate one at the permissive temperature and the other at the nonpermissive temperature. 9 Identify mutant colonies and test their ability to replicate their DNA when shifted to the nonpermissive temperature. E. coli has many vital genes that are not involved in DNA replication; so, only a subset of ts mutants would carry mutations affecting the replication process. Therefore, researchers in the 1960s had to screen thousands of ts mutants to get to those involved in DNA replication; this is sometimes called a “brute force” genetic screen. Table 11.3 summarizes some of the genes that were identified using this strategy. The isolation of DNA mutants was important in several ways: It allowed for the identification of the proteins that were defective in the mutant. It allowed for the mapping of these mutations along the E. coli chromosome. It provided an important starting point for the subsequent cloning and sequencing of these genes. Eukaryotic DNA Replication multiple origins of replication along t he dna, until two molecules of dna t hat are identical t o one another and identical to what we started with Eukaryotic DNA replication is not as well understood as bacterial replication. The two processes do have extensive similarities; the bacterial enzymes described in Table 11.1 have also been found in eukaryotes. Nevertheless, DNA replication in eukaryotes is more complex given: Large linear chromosomes. Tight packaging within nucleosomes. More complicated cell cycle regulation. Initiation occurs at multiple origins of replication on linear eukaryotic chromosomes. Eukaryotes have long linear chromosomes; therefore, they require multiple origins of replication to ensure that the DNA can be replicated in a reasonable time. DNA replication proceeds bidirectionally from many origins of replication (see fig. 11.21). 10 more differences than t here are sim. origins of replication in eukaryotic or ARS they are the same thing does not apply to OriC have kind of a smiilar f unction not necessarily sequence The origins of replication found in eukaryotes have some similarities to those of bacteria. Origins of replication in Saccharomyces cerevisiae are termed ARS (Autonomously Replicating Sequence) elements: They are 100-150 bp in length. They have a high percentage of A and T. They have three or four copies of a specific sequence (similar to the bacterial DnaA boxes). Replication begins with assembly of the prereplication complex (preRC) composed of at least 14 different proteins; an important part of this is the origin recognition complex (ORC), a six-subunit complex that acts as the initiator of eukaryotic DNA replication. Other preRC proteins bind, including MCM helicase. Binding of MCM completes DNA replication licensing; the origin is capable of initiating DNA synthesis. Binding of at least 22 additional proteins is required to initiate synthesis during S phase. starts out as ORC, t hen preRC proteins c ome along and get t hings started starts in G1 . Eukaryotes contain several different DNA polymerases. alpha is involved in making the primer...before we just had primase, it attached and made a short rna primer once primer is made (say leading strand), \dna pol aplha and primase come off and there is an exchange and dna eps attaches to leading s trand, stays on there s ynth 5-3 along leading s trand meanwhile, at lagging, rna/dna primer comes off and dna pol delta comes on and makes an okazaki f ragment then another primer is being made by dna aplha t hen another dna delta makes another OF Mammalian cells contain well over a dozen different DNA polymerases (see table 11.4). Four, alpha ("), delta (#), epsilon ($) and gamma (%), have the primary function of replicating DNA: ", # and $ ! Nuclear DNA % ! Mitochondrial DNA DNA polymerase " is the only polymerase to associate with primase. The DNA polymerase "/primase complex synthesizes a short RNA-DNA hybrid – 10 RNA nucleotides followed by 20 to 30 DNA nucleotides. This is used by DNA polymerase # or $ for the processive elongation of the leading and lagging strands; current evidence suggests that DNA polymerase ! may be involved with leading strand synthesis, while DNA polymerase " has a greater role in lagging-strand synthesis. The exchange of DNA polymerase " for # or $ is called a polymerase switch; it occurs only after the RNA-DNA hybrid is made (see fig. 11.22). 11 dna polymerase delta plows right through and k nocks off primer of t emplate strand, and t hen the fl ap thats f ormed is chewed up by fl ap endonucelase DNA pol B kind of f unctions like DNA polymerases of Bacterial Replication DNA polymerases also play a role in DNA repair. DNA polymerase ! is not involved in DNA replication; it plays a role in baseexcision repair, removal of incorrect bases from damaged DNA. Recently, more DNA polymerases have been identified. Lesion-replicating polymerases are involved in the replication of damaged DNA; they can synthesize a complementary strand over the abnormal region. telomeres are short The s equences ends of eukaryotic chromosomes are replicated by telomerase. Linear eukaryotic chromosomes have telomeres at both ends; the term telomere refers to the complex of telomeric DNA sequences and bound proteins. Telomeric sequences consist of moderately repetitive tandem arrays and a 3’ overhang that is 12-16 nucleotides long. Telomeric sequences typically consist of several guanine nucleotides and often many thymine nucleotides (see table 11.5). DNA polymerases possess two unusual features: They synthesize DNA only in the 5’ to 3’ direction. TELOMERIC = GT have overhangs at the end, every progressive round of dna replication gets shorter and shorter and shorter. except in sex cells. our telomeres get depleted as we get older They cannot initiate DNA synthesis. These two features pose a problem at the 3’ end of linear chromosomes – the end of the strand cannot be replicated. If this problem is not solved, the linear chromosome becomes progressively shorter with each round of DNA replication. The cell solves this problem by adding DNA sequences to the ends of telomeres; this requires a specialized mechanism catalyzed by the enzyme telomerase. Telomerase contains protein and RNA. The RNA is complementary to the DNA sequence found in the telomeric repeat. This allows the telomerase to bind to the 3’ overhang. The lengthening mechanism is outlined in figure 11.25. Please see the Conceptual Summary and Experimental Summary for Chapter 11 on page 292. This lecture outline was prepared from Genetics: Analysis and Principles, by Brooker, 2009 (3rd edition). It contains phrases and entire sentences taken verbatim from that source, and is in no way meant to represent original work by Mark Bierner. 12 ...
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