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ADN Replication

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Nguồn: Internet
Người gửi: Phan Thanh Quyền (trang riêng)
Ngày gửi: 09h:02' 19-04-2008
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Chapter 11 : DNA Replication

Outline: * Semiconservative Replication o Meselson-Stahl Experiment * DNA polymerases and DNA elongation * Molecular model of DNA replication o Initiation of Replication o Semidiscontinuous DNA replication o Rolling circle replication * Replication of telomeres in eukaryotes DNA replication underlies the process of inheritance at all levels (cellular, organismal, population). DNA replication occurs as prelude to cell division ( S phase of cell cycle in eukaryotes). DNA in all organisms is the end point in a continuous series of replications going back to the origin of life, almost 4 billion yrs ago. DNA replication is based on complementarity of DNA molecules and on ability of proteins to form specific interactions with specific sequences of DNA. Semiconservative Replication  * Watson and Crick model of DNA suggested that each strand could serve as template for synthesis of new strand. Their model is called semiconservative DNA replication * Two other models based on template-based synthesis were also proposed by others (Fig 11.1): o Conservative model: parental strands rejoin after they are used as templates, resulting in two DNA moleucles, one made of two parental strands, and the other made entirely of newly synthesized DNA. o Dispersive model: parental DNA cleaved into DNA segments that act as templates for the synthesis of new DNA and then somehow segments reassemble into double stranded DNA made of parental and progeny DNA which are interspersed. o All three models made different predictions about the nature of DNA after one and two rounds of replication (Fig 11.1). Meselson-Stahl Experiment * Meselson and Stahl (1958) used a heavy isotope of nitrogen (15N) and equilibrium density gradient centrifucation to show that DNA replicated in semiconservative manner in E. coli (Fig11.2). o grew E. coli for many generations in medium containing 15NH4Cl (15N is a heavier isotope than 14N). This resulted in DNA containing 15N instead of 14N. 15N DNA can be seperated from 14N DNA by ultracentifugation in a CsCl gradient. o 15N-labeled bacteria were then transferred to medium containing 14N and allowed to grow for several generations, and sampled after each replication cycle. o After one generation in 14N, all the DNA had a density intermediate from 15N-DNA and 14N-DNA, just as predicted by the semiconservative and dispersive models. + this result ruled out the conservative model because it predicted that there should be two bands (one containing light DNA and the other heavy DNA). o To distinguish between the semiconservative model and the dispersive model, E.coli were grown for another generation. Two bands were observed, as expected by the semiconservative model. The dispersive model predicted that there should only be one band, therefore it was also ruled out. The results were all consistent with the semiconservative model. o * Semiconservative DNA replication also occurs in eukaryotes (see harlequin chromosomes in Fig 11.3). DNA polymerases and DNA elongation * In 1955, Arthur Korberg identified the first DNA polymerase (DNA Pol I). Initially it was thought to be the main DNA replication enzyme, but mutant E.coli defective in the gene encoding for DNA pol I divided normally, indicating that there must be other enzymes involved. * Five DNA polymerases have now been identified in E. coli. DNA Pol II, IV, and V are involved in DNA repair. DNA pol I and III are involved in DNA replication. * All DNA polymerases catalyze the polymerization of nucleotide precursors (dNTPs) into a DNA chain . The reaction is shown in Fig 11.4 and has three main features: 1. DNA pols catalyze the formation of a phosphodiester bond between the 3`-OH group of the deoxyribose on the last nucleotide in the chain and the 5`-phosphate of the incoming nucleotide. The energy is supplied by the hydrolysis of the two phosphates from the dNTP. All DNA polymerases require a primer (i.e they can not add the first nucleotide ). 2. DNA polymerases require a template. The particular nucleotide added depends on correct complementary base pairing with the template. DNA pols are fast. In E. coli, DNA pol I and II can polymerize ~ 850 nt per sec. In humans, its a lot slower (60-90 nt/sec). 3. All DNA polymerases synthesize DNA in the 5` to 3` direction. * DNA pol I and II also have exonuclease activity. o DNA pol I and III have 3`-> 5` exonuclease activity. This is a proofreading mechanism. DNA pols add an incorrect base with a frequency of 10-6. When an incorrect base is added, the enzyme detects that it made a mistake, and uses its 3` to 5` exonuclease activity to move back and remove the incorrect base. With proofreading, the error rate drops to 10-9. o DNA pol I also has 5` -> 3` exonuclease activity. This allows it to remove DNA or RNA from the 5` end of a moleecule. This is essential during DNA replication of the lagging strand. Model of DNA Replication in E. coli * The bare-bone mechanics of DNA replication is similar in all organisms. However, we will only focus on DNA replication in E. coli, where it is best understood. Along the way, significant differences between prokaryotic and eukaryotic DNA replication will be highlighted. * Basic research into the mechanisms of DNA replication in E. coli (as well as transcription and translation) has led to the identification and cloning of dozens of genes involved in these processes (Table 11.1). The creative use of these gene products has given us a tremendous power to manipulate genes and genomes according to our will. Initiation of Replication * Initiation of replication starts at a DNA sequence called the replicator, which includes the origin of replication (OriC) (AT-rich) where DNA is denatured into single strands to form a replication bubble. At either end of a bubble there is a replication fork, where DNA synthesis occurs, using each separated strand as a template. o Circular genomes of prokaryotes contain a single origin of replication. o In eukaryotes, linear chromosomes contain many origins of replication (allows faster replication). o Synthesis proceeds bidirectionaly at replication fork. Eventually, replicated double helices join each other, producing two daughter molecules (Fig 11.9)(sister chromatids, in eukaryotes) * Initiation of replication starts with the binding of an initiator protein which denatures the oriC and then recruits a DNA helicase (one for each strand) which untwists the DNA in both directions (energy comes from hydrolysis of ATP) (Fig 11.5). * Next, each helicase recruits a DNA primase to form a primosome. DNA primase makes the necessary RNA primers ( 5-10 nts) needed by DNA polymerase III. * The next step involves the assembly of the rest of the proteins involved in DNA replication. These proteins associate to form a replisome. There is a replisome at each replication fork.  Semidiscontinuous DNA replication * The replication steps are identical at each replication fork, so we focus on just one. The entire process is shown in Fig 11.6. * After the helicase unwinds the DNA, the single stranded DNA is prevented from reannealing by binding to single-strand DNA-binding proteins (SSBs) (about 200 /rep fork). * DNA pol III dimer (part of replisome) now initiates polymerization by adding dNTPs to the RNA primer on each of the strands. Because strands in double helix are in antiparallel configuration, and DNA polymerases add dNTPs in 5` to 3` direction, the two strands are synthesized differently: o Leading strand synthesized continuously; only one primer required; DNA pol III moves in same direction as replication fork. o Lagging strand synthesized discontinuously as Okazaki fragments, which are later ligated by DNA ligase. Each Okazaki fragment requires a primer. DNA pol III moves in opposite direction to replication fork.  * In Lagging-strand synthesis, DNA Pol III ends polymerization when it encounters double stranded DNA ahead (from previous Okazaki fragment). It dissociates from the DNA, leaving a gap in one strand. This gap is recognized as damaged DNA and is repaired by DNA Pol I. * DNA Pol I removes primers and fills in gaps (has 5`-3` exonuclease activity).

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 * DNA ligase joins 3` end of one Okazaki fragment to 5` end of downstream Okazaki fragment (Fig 11.7). * As helicase unwinds DNA ahead of replication fork, positive supercoils form elsewhere in the molecule. For replication fork to move, the helix must rotate (estimated at 50 revolutions/sec). The problem of supercoiling is solved by the action of topoisomerases (specifically a Gyrase) which introduce negative supercoils to counteract positive supercoils intoduce by helicases. Rolling circle replication * For many viral DNAs and some plasmids (e.g. F plasmid in E. coli), rolling circle replication has been demonstrated.

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 * Synthesis usually continues beyond a single chromosomal unit. This results in many head-to-tail copies of the plasmid, which is then cut and rejoined into new circular molecules. Replication of telomeres in eukaryotes * There are special problems associated with replication of the ends of linear chromosomes (called telomeres). Recall that DNA polymerases only add nucleotides to the 3` end of a growing chain. When the linear chromosomes of eukaryotes replicate, the resulting daughter molecules will each have an RNA primer left over at the 5`end (Fig 11.14). This RNA primer is removed, leaving a single stranded DNA segment. If not fixed, this single-stranded DNA region will get degraded, and the linear chromosomes will get shorter with each round of DNA replication. * In most eukaryotes, an enzyme called telomerase, maintains the ends of chromosomes by adding telomere repeats to chromosom
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