ADN Replication

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). This image has been resized. Click this bar to view the full image. The original image is sized 720x540 and weights 49KB. * 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. This image has been resized. Click this bar to view the full image. The original image is sized 690x499 and weights 79KB. * 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 chromosome ends. The mechanism is shown below (and in Fig 11.5). * Telomerase is a ribonucleoprotein (has RNA molecule as part of its structure) which adds tandem repeats to the 3' end of chromosomes using an RNA molecule as a template. After is has added many telomeric repeats and has left, a new DNA molecule is made starting from a new RNA primer, which is again is removed, but by this time the chromosme has already been extended. * The absence of telomerase activity in cells is correlated with senescence of cells (i.e. die after certain number of cell divisions). Conversely, enhanced telomerase activity correlated with cell immortalily (i.e. cells divide indefinately). o cells with short telomerse undergo fewer doublings than ones with long telomerase. o fibroblasts form individuals with progeria (rare disease characterized by premature aging) have short telomeres. o most somatic cells have no active telomerase (divide only 20-60 times) o sperm cells, stem cells and unicellular eukaryotes (essentially immortal ) have active telomerase and stable telomeres. o cancer cells, which are also essentially immortal, have active telomerase (promising target for drug design) o Elimination of telomerase activity in somatic cells may be a cellular senescence mechanism that protects multicellular organisms from cancer Chapter 13 : Transcription * Outline o Genes and RNA o Properties of RNA o Classes of RNA o Making functional transcripts + RNA polymerases + Initiation + Elongation + Termination o RNA processing in eukaryotes Genes and RNA Biological information flow from DNA to protein requires an RNA intermediate. RNA is produced by a process that copies the nucleotide sequence in DNA to produce a transcript. This process is called transcription. Properties of RNA 1. Single stranded, but can undergo intramolecular base-pairing - forms variety of 3D structures specified by sequence. 2. Ribose sugar (not deoxyribose) 3. Uracyl in place of thymine Classes of RNA * There are a variety of different RNAs that can be classified into two classes. o 1. Informational RNAs (e.g. messenger RNA) + intermediate which is later translated into protein. + most genes encode mRNA o 2. Functional RNAs + never translated + diverse roles in cell + main classes of functional RNAs play critical roles in various steps in the information processing of DNA to protein: # rRNA - components of ribosome # tRNA - bring amino acids to mRNA during translation # snRNA (small nucleolar RNAs) - involved in splicing of introns # scRNAs (small cytoplasmic RNAs) - protein trafficking * All DNA and RNA function is based on two key elements: o 1. Complementary bases in single stranded nucleotide chains can H-bond to form double stranded structures. o 2. Specific sequences can be recognized by specific nucleic-acid binding proteins. Making functional transcripts * Transcription uses one DNA strand as template o Strands of double helix must be separated, so that one of these strands (template strand) can serve as template to direct the synthesis of transcript. * Either strand along the chromosome can serve as template, but for a given gene, its always the same strand. * RNA polymerase catalyzes the synthesis of RNA using DNA template (Fig 13.1). o RNA grows in 5' to 3' direction, and the template is read in the 3' to 5' direction. o sequence of RNA is complementary to template strand (noncoding strand), but the same as nontemplate strand (coding strand) except T replaced with U. * A typical prokaryotic gene has the folowing features: RNA Polymerases * Prokaryotes have only one RNA Polymerase but eukaryotes have 3: 1. RNA Pol I: transcribes rRNA genes 2. RNA Pol II: transcribes protein encoding genes 3. RNA Pol III: transcribes other functional RNAs (tRNAs, snoRNAs etc .) * In eukaryotes, transription takes place in nucleus. * In prokaryotes, transcription and translation are coupled. * Transcription involves 3 distinct stages: initiation, elongation, and termination. Initiation * In E. coli, transcription requires a complex of RNA polymerase and the sigma factor (s) which binds to a promoter. The RNA polymerase core enzyme (4 has four subunits, two a, one b and one b') complexed with the sigma factor is known as the holoenzyme. Once transcription is initiated, the sigma factor dissociates. * promoter = DNA sequence to which RNA Pol binds to initiate transcription. o note that by convention, gene is labelled the same way as RNA transcript. So promoter is at 5' end of gene (Fig 13.3). * RNA pol + sigma factor scans DNA for promoter sequence, binds DNA at the promoter sequence (- 10 region and -35 region), unwinds it, and begins synthesis of a transcript at transcription initiation site. Promoter sequences are not transcribed. NOTE: RNA pol does not need a primer to initiate RNA synthesis not does it need a helicase. o there are consensus sequences for all promoters in E. coli. A consensus sequence is the sequence found most frequently at each position. E.g consensus sequence at -10 position is 5'-TATAAT-3' o The more similar the promoter sequence is to the consensus, the higher the rate of transcription. o It is the sigma factor that binds the promoter. Different sigma factors bind different promoters. * What is described above is the minimum required for transcription initiation. In chapter 19 we will study how genes are regulated in prokaryotes in more detail. Elongation * RNA pol moves along DNA, maintaining transcription "bubble" to expose template strand, and catalyzes the 3' elongation of transcript. o energy for reaction derived from splitting high-energy triphosphates into monophosphates. o rate of transcription is about 30-50 nt/sec Termination * Results from different mechanisms signalled by termination sequences at 3' end of a gene. Two mechanisms known: o Rho-independent termination + involves formation of hairpin loop (Fig 13.5) in nascent transcript causing RNA strand and RNA Pol to be released from DNA template. o Rho-dependent termination [...]... scientists that DNA was the genetic material * Hershey and Chase studied bacteriophage T2, which was known to be made entirely of protein and DNA T2 phage replicates by invading E coli, taking over its replication machinery to make progeny phage The host cell then lyses and releases progeny phage capable of infecting new cells * Their experiment started with the radioactive-labeling of either DNA (with... with guanine Because of complementary base pairing, the sequence of one strand can be inferred from the other o important that strands be held by weak bonds, so that DNA strands can be separated during replication and transcription 5 Base pairs are 0.34 nm apart A complete turn of the helix takes 3.4 nm, and the diameter of the helix is a uniform 2 nm 6 Double helix contains a major and a minor groove... Plasmids are occasionally found in fungal and plant cells: o most are inside mitochindria or chloroplast o unlike in prokaryotes, provide no benefit to host * Plasmids rely on cellular machinery of host for replication and maintenance * Plasmids are very useful in recombinant DNA technology Organellar DNA * Both mitochondria and chroloplasts are derived from once free-living bacteria which became endosymbionts... have a 12 nt single stranded regions that are complementary # when lambda infects E coli, the sticky ends base pair and chromosome becomes circular Chromosome reverts back to linear form during phage replication and packaging Eubacterial Genomes * Genome size is relatively small (average is 4 Mb) , but there's considerable variation o Bacillus megasterium = 30 Mb o Yeast = 12.1 Mb * In most cases,... spindle fibers; responsible for proper segregation of chromsomes during cell division o made up of highly repetitive DNA * Telomeres o telomeres are repetitive sequences at ends of chromosomes required for replication and chromosome stability o characteristically heterochromatic Chapter 1: Introduction * Outline o Introduction o Classical and Molecular Genetics o Important Concepts of Genetics + DNA, Genes . Initiation of Replication o Semidiscontinuous DNA replication o Rolling circle replication * Replication of telomeres in eukaryotes DNA replication underlies. DNA replication. These proteins associate to form a replisome. There is a replisome at each replication fork. Semidiscontinuous DNA replication * The replication