Download Lecture Notes for DNA Replication | BIO 325 and more Study notes Genetics in PDF only on Docsity!
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 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 Dispersive model: parental and daughter DNA isb 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) The starting material is a strain of E. coli that has been grown for many generations in the presence of 15 N; therefore, all of the nitrogen in the DNA is labeled with 15 N Add an excess of 14 N-containing compounds to the bacterial cells so that all of the newly made DNA will contain 14 N Incubate the cells for various lengths of time
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 The data (see fig. 11.3) Interpreting the data 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) 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
Details of DNA synthesis 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 right hand Template DNA threads through the palm 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 discoverers
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 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 β subunit is in the shape of a ring; it is termed the clamp protein γ subunit is needed for β to initially clamp onto the DNA; it is termed the clamp- loader protein δ, δ’ and ψ subunits are needed for the optimal function of the α and β subunits The effect of processivity is quite remarkable In the absence of the β 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 β 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 T and T2; T1 stops counterclockwise forks, T2 stops clockwise forks
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 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
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 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 32 P-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
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 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 In 1968, Huberman and Riggs provided evidence for the multiple origins of replication (see fig. 11.20) DNA replication proceeds bidirectionally from many origins of replication (see fig. 11.21)
The origins of replication found in eukaryotes have some similarities to those of bacteria Origins of replication in Saccharomyces cerevisiae are termed ARS elements (Autonomously Replicating Sequence) 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 Eukaryotes contain several different DNA polymerases Mammalian cells contain well over a dozen different DNA polymerases (see table 11.4) Four, alpha (α), delta (δ), epsilon (ε) and gamma (γ), have the primary function of replicating DNA α, δ and ε Nuclear DNA γ Mitochondrial DNA DNA polymerase α is the only polymerase to associate with primase The DNA polymerase α/primase complex synthesizes a short RNA-DNA hybrid – 10 RNA nucleotides followed by 20 to 30 DNA nucleotides This is used by DNA polymerase δ or ε for the processive elongation of the leading and lagging strands; current evidence suggests a greater role for DNA polymerase δ The exchange of DNA polymerase α for δ or ε is called a polymerase switch; it occurs only after the RNA-DNA hybrid is made (see fig. 11.22)
