分子生物学 第十三章 DNA复制（DNA replication）.ppt

Chapter 13,DNA replication,13.1 Introduction13.2 DNA polymerases are the enzymes that make DNA13.3 DNA synthesis is semidiscontinuous 13.4 Coordinating synthesis of the lagging and leading strands 13.5 The replication apparatus of phage T4 13.6 Creating the replication forks at an origin 13.7 Common events in priming replication at the origin13.8 Does methylation at the origin regulate initiation?13.9 Licensing factor controls eukaryotic rereplication,Replisome is the multiprotein structure that assembles at the bacterial replicating fork to undertake synthesis of DNA. Contains DNA polymerase and other enzymes.,13.1 Introduction,DNA polymerases are enzymes that synthesize a daughter strand(s) of DNA (under direction from a DNA template). May be involved in repair or replication.DNA replicase is a DNA-synthesizing enzyme required specifically for replication.Repair of damaged DNA can take place by repair synthesis, when a strand that has been damaged is excised and replaced by the synthesis of a new stretch. It can also take place by recombination reactions, when the duplex region containing the damaged is replaced by an undamaged region from another copy of the genome.Replication of duplex DNA takes place by synthesis of two new strands that are complementary to the parental strands. The parental duplex is replaced by two identical daughter duplexes, each of which has one parental strand and one newly synthesized strand. It is called semiconservative because the conserved units are the single strands of the parental duplex.,13.2 DNA polymerases are the enzymes that make DNA,Figure 13.1 Semiconservative replication synthesizes two new strands of DNA.,13.2 DNA polymerases are the enzymes that make DNA,Figure 13.2 Repair synthesis replaces a damaged strand of DNA.,13.2 DNA polymerases are the enzymes that make DNA,Figure 13.3 DNA synthesis occurs by adding nucleotides to the 3-OH end of the growing chain, so that the new chain is synthesized in the 5-3 direction. The precursor for DNA synthesis is a nucleoside triphosphate, which loses the terminal two phosphate groups in the reaction.,13.2 DNA polymerases are the enzymes that make DNA,Figure 13.4 Only one DNA polymerase is the replicase. The others participate in repair of damaged DNA.,13.2 DNA polymerases are the enzymes that make DNA,Nick translation describes the ability of E. coli DNA polymerase I to use a nick as a starting point from which one strand of a duplex DNA can be degraded and replaced by resynthesis of new material; is used to introduce radioactively labeled nucleotides into DNA in vitro.,13.3 DNA polymerases have various nuclease activities,Proofreading refers to any mechanism for correcting errors in protein or nucleic acid synthesis that involves scrutiny of individual units after they have been added to the chain.,13.4 DNA polymerases control the fidelity of replication,Figure 13.6 Bacterial DNA polymerases scrutinize the base pair at the end of the growing chain and excise the nucleotide added in the case of a misfit.,13.4 DNA polymerases control the fidelity of replication,Figure 13.5 Nick translation replaces part of a pre-existing strand of duplex DNA with newly synthesized material.,13.3 DNA polymerases have various nuclease activities,13.3 DNA polymerases have various nuclease activities,13.3 DNA polymerases have various nuclease activities,Figure 13.12 There are several methods for providing the free 3-OH end that DNA polymerases require to initiate DNA synthesis.,13.8 Priming is required to start DNA synthesis,Figure 13.7 Crystal structure of phage T7 DNA polymerase has a right hand structure. DNA lies across the palm and is held by the fingers and thumb. Photograph kindly provided by Charles Richardson and Tom Ellenberger.,13.5 Some DNA polymerases have a common structure,Figure 13.8 The catalytic domain of a DNA polymerase has a DNA-binding cleft created by three subdomains. The active site is in the palm. Proofreading is provided by a separate active site in an exonuclease domain.,13.5 Some DNA polymerases have a common structure,Lagging strand of DNA must grow overall in the 3-5 direction and is synthesized discontinuously in the form of short fragments (5-3) that are later connected covalently.Leading strand of DNA is synthesized continuously in the 5-3 direction.Okazaki fragments are the short stretches of 1000-2000 bases produced during discontinuous replication; they are later joined into a covalently intact strand.Semidiscontinuous replication is mode in which one new strand is synthesized continuously while the other is synthesized discontinuously.,13.6 DNA synthesis is semidiscontinuous,Figure 13.9 The leading strand is synthesized continuously while the lagging strand is synthesized discontinuously.,13.6 DNA synthesis is semidiscontinuous,SSB is the single-strand protein of E. coli, a protein that binds to single-stranded DNA.,13.7 Single-stranded DNA is needed for replication,Figure 13.22 Synthesis of Okazaki fragments requires priming, extension, removal of RNA, gap filling, and nick ligation.,13.10 Coordinating synthesis of the lagging and leading strands,Figure 13.23 DNA ligase seals nicks between adjacent nucleotides by employing an enzyme-AMP intermediate.,13.10 Coordinating synthesis of the lagging and leading strands,Figure 13.10 fX174 DNA can be separated into single strands by the combined effects of 3 functions: nicking with A protein, unwinding by Rep, and single-strand stabilization by SSB.,13.7 Single-stranded DNA is needed for replication,Figure 13.13 Initiation requires several enzymatic activities, including helicases, single-strand binding proteins, andsynthesis of the primer.,13.8 Priming is required to start DNA synthesis,Figure 13.13 Initiation requires several enzymatic activities, including helicases, single-strand binding proteins, andsynthesis of the primer.,13.8 Priming is required to start DNA synthesis,Figure 13.12 Leading and lagging strand polymerases move apart.,13.8 Priming is required to start DNA synthesis,Figure 13.18 DNA polymerase III holoenzyme assembles in stages, generating an enzyme complex that synthesizes the DNA of both new strands.,13.10 Coordinating synthesis of the lagging and leading strands,Figure 13.19 The b subunit of DNA polymerase III holoenzyme consists of a head to tail dimer (the two subunits are shown in red and orange) that forms a ring completely surrounding a DNA duplex (shown in the center). Photograph kindly provided by John Kuriyan.,13.10 Coordinating synthesis of the lagging and leading strands,Figure 13.20 Each catalytic core of Pol III synthesizes a daughter strand. DnaB is responsible for forward movement at the replication fork.,13.10 Coordinating synthesis of the lagging and leading strands,Figure 13.21 Core polymerase and the b clamp dissociate at completion of Okazaki fragment synthesis and reassociate at the beginning.,13.10 Coordinating synthesis of the lagging and leading strands,Figure 13.16 Tus binds to ter asymmetrically and blocks replication in only one direction.,13.9 The primosome is needed to restart replication,Figure 13.24 Similar functions are required at all replication forks.,13.11 The replication apparatus of phage T4,Figure 13.24 Similar functions are required at all replication forks.,13.11 The replication apparatus of phage T4,Figure 13.25 The minimal origin is defined by the distance between the outside members of the 13-mer and 9-mer repeats.,13.12 Creating the replication forks at an origin,Figure 13.26 Prepriming involves formation of a complex by sequential association of proteins, leading to the separation of DNA strands.,13.12 Creating the replication forks at an origin,Figure 13.27 The complex at oriC can be detected by electron microscopy. Both complexes were visualized with antibodies against DnaB protein. Photographs kindly provided by Barbara Funnell.,13.12 Creating the replication forks at an origin,13.13 Common events in priming replication at the origin,Figure 13.28 Transcription initiating at PR is required to activate the origin of lambda DNA.,Figure 13.29 The lambda origin for replication comprises two regions. Early events are catalyzed by O protein, which binds to a series of 4 sites; then DNA is melted in the adjacent A-T-rich region. Although the DNA is drawn as a straight duplex, it is actually bent at the origin.,13.13 Common events in priming replication at the origin,13.13 Common events in priming replication at the origin,13.14 Does methylation at the origin regulate initiation?,Figure 13.30 Replication of methylated DNA gives hemimethylated DNA, which maintains its state at GATC sites until the Dam methylase restores the fully methylated condition.,13.14 Does methylation at the origin regulate initiation?,Figure 13.31 Only fully methylated origins can initiate replication; hemimethylated daughter origins cannot be used again until they have been restored to the fully methylated state.,13.14 Does methylation at the origin regulate initiation?,Figure 13.32 A membrane-bound inhibitor binds to hemimethylated DNA at the origin, and may function by preventing the binding of DnaA. It is released when the DNA is remethylated.,13.15 Licensing factor controls eukaryotic rereplication,Figure 13.33 A nucleus injected into a Xenopus egg can replicate only once unless the nuclear membrane is permeabilized to allow subsequent replication cycles.,13.15 Licensing factor controls eukaryotic rereplication,Figure 13.34 Licensing factor in the nucleus is inactivated after replication. A new supply of licensing factor can enter only when the nuclear membrane breaks down at mitosis.,13.15 Licensing factor controls eukaryotic rereplication,Figure 13.35 Proteins at the origin control susceptibility to initiation.,13.16 Summary,DNA synthesis occurs by semidiscontinuous replication, in which the leading strand of DNA growing 53 is extended continuously, but the lagging strand that grows overall in the opposite 35 direction is made as short Okazaki fragments, eachsynthesized 53. The leading strand and each Okazaki fragment of the lagging strand initiate with an RNA primer that is extended by DNA polymerase. Bacteria and eukaryotes each possess more than one DNA polymerase activity. DNA polymerase III synthesizes both lagging and leading strands in E. coli. Many proteins are required for DNA polymerase III action and several constitute part of the replisome within which it functions.,Figure 12.16 The rolling circle generates a multimeric single-stranded tail.,13.7 Single-stranded DNA is needed for replication,Primer is a short sequence (often of RNA) that is paired with one strand of DNA and provides a free 3-OH end at which a DNA polymerase starts synthesis of a deoxyribonucleotide chain.,13.8 Priming is required to start DNA synthesis,Figure 13.11 A DNA polymerase requires a 3-OH end to initiate replication.,13.8 Priming is required to start DNA synthesis,Figure 12.34 Replication of ColE1 DNA is initiated by cleaving the primer RNA to generate a 3-OH end. The primer forms a persistent hybrid in the origin region.,13.8 Priming is required to start DNA synthesis,Figure 12.16 The rolling circle generates a multimeric single-stranded tail.,13.8 Priming is required to start DNA synthesis,Figure 13.5 Nick translation replaces part of a pre-existing strand of duplex DNA with newly synthesized material.,13.8 Priming is required to start DNA synthesis,Figure 12.15 Adenovirus terminal protein binds to the 5 end of DNA and provides a C-OH end to prime synthesis of a new DNA strand.,13.8 Priming is required to start DNA synthesis,Primosome describes the complex of proteins involved in the priming action that initiates replication on fX-type origins. It is also involved in restarting stalled replication forks.,13.9 The primosome is needed to restart replication,Figure 13.14 Replication is halted by a damaged base or nick in DNA.,13.9 The primosome is needed to restart replication,Figure 14.35 An E. coli retrieval system uses a normal strand of DNA to replace the gap left in a newly synthesized strand opposite a site of unrepaired damage.,13.9 The primosome is needed to restart replication,Figure 13.15 The primosome is required to restart a stalled replication fork after the DNA has been repaired.,13.9 The primosome is needed to restart replication,Figure 12.7 Replication termini in E. coli are located beyond the point at which the replication forks actually meet.,13.9 The primosome is needed to restart replication,Figure 13.17 Leading and lagging strand polymerases move apart.,13.10 Coordinating synthesis of the lagging and leading strands,Figure 13.26 Prepriming involves formation of a complex by sequential association of proteins, leading to the separation of DNA strands.,13.13 Common events in priming replication at the origin,13.14 Does methylation at the origin regulate initiation?,Figure 12.26 Attachment of bacterial DNA to the membrane could provide a mechanism for segregation.,13.15 Licensing factor controls eukaryotic rereplication,Figure 1.10 Replication of DNA is semiconservative.,13.15 Licensing factor controls eukaryotic rereplication,Figure 12.10 An ARS extends for 50 bp and includes a consensus sequence (A) and additional elements (B1-B3).,13.16 Summary,DNA synthesis occurs by semidiscontinuous replication, in which the leading strand of DNA growing 53 is extended continuously, but the lagging strand that grows overall in the opposite 35 direction is made as short Okazaki fragments, eachsynthesized 53. The leading strand and each Okazaki fragment of the lagging strand initiate with an RNA primer that is extended by DNA polymerase. Bacteria and eukaryotes each possess more than one DNA polymerase activity. DNA polymerase III synthesizes both lagging and leading strands in E. coli. Many proteins are required for DNA polymerase III action and several constitute part of the replisome within which it functions.,The replisome contains an asymmetric dimer of DNA polymerase III; each new DNA strand is synthesized by a different core complex containing a catalytic () subunit. Processivity of the core complex is maintained by the clamp, which forms a ring round DNA. The looping model for the replication fork proposes that, as one half of the dimer advances to synthesize the leading strand, the other half of the dimer pulls DNA through as a single loop that provides the template for the lagging strand. The transition from completion of one Okazaki fragment to the start of the next requires the lagging strand catalytic subunit to dissociate from DNA and then to reattach to a clamp at the priming site for the next Okazaki fragment.,DnaB provides the helicase activity at a replication fork; this depends on ATP cleavage. DnaB may function by itself in oriC replicons to provide primosome activity by interacting periodically with DnaG, which provides the primase that synthesizes RNA.,Phage T4 codes for a sizeable replication apparatus, consisting