Chapter 03: DNA Replication,3.1 The principle of DNA replication 3.2 DNA replication model 3.3 Enzymes and protein needed in DNA replication 3.4 Process of DNA replication 3.5 Telomere and Telomerase,3.1 The principle of DNA replication,3.1.1 semi-conservative replication Definition: during replication, the two parental strands separate and each acts as a template to direct the synthesis of a new complementary daughter strand. DNA replication pattern: conservative model、semi-conservative model、dispersive model,E.coli ：Radioactive isotope(15N) labelCsCl density gradient centrifugation,Importance: the stability of DNA metabolismThe stability is relative, while the variation is absolute,OK !,How ?,3.1.2 Semi-discontinuous replication,Replication fork,1968 Okazaki radioactive label (3H-dTTP) + CsCl density gradient centrifugation label enters newly synthesized DNA in the form of short fragments(1000-2000 bases) The ligase temperature-sensitive mutants under the temperature in which the ligase is inactive a large amount of short fragments but no lagging strand Conclusion: The lagging strand must be synthesized in the form of Okazaki fragments,On the leading strand, DNA synthesis can proceed continuously in the 5 to 3 direction as the parental duplex is unwound. On the lagging strand, a stretch of single-stranded parental DNA must be exposed, and then a segment is synthesized in the reverse direction (relative to fork movement). A series of these fragments are synthesized, then they are joined together to create an intact lagging strand.,Okazaki fragments: the short stretches of 1000-2000 bases in prokaryotes (100-200 bases in eukaryotes) produced during semi-discontinuous replication, they are later joined into a covalently intact strand. Semi-discontinuous replication- the leading strand is synthesized continuously while the lagging strand is synthesized discontinuously,3.1.3 Primer,A common feature of all DNA polymerases is that they cannot initiate synthesis of a DNA chain de novo. All DNA polymerases require a 3-OH end to initiate DNA synthesis. For DNA replication, a special RNA polymerase called a primase synthesizes an RNA chain(primer) that provides the priming end.,ssDNA virus,RF（replicating form）,No M13 RF,Rifampin,M13,Rifampin,M13 RF,M13 RF,M13,Rifampin,M13,expression, packaging, release,Its necessary of RNA polymerase to synthesize a RNA in the formation of M13 RF；,after the initiation of M13 RF, the inhibition of rifampin is invalid,Conclusion,The first evidence supporting RNA primer,Rifampin is inhibitor of E.coli RNA polymerase,Purpose: keep the high “faithfulness“ Reason: DNA polymerase has proofreading activity, while RNA polymerase dont. After the low-fidelity primer complete its function, it will be replaced by high-fidelity DNA synthesized DNA polymerase. Results: improve the accuracy of the DNA replication up to 105 times.,UnidirectionalBidirectional (more common),3.1.4 Direction,Autoradiography, Bacillus sp.,Gyurasits and Wake,3.2 Replication Model,Replicon：means a sequence from a origin to a terminus. Origins tend to be AT-rich to make opening easier,All prokaryotic chromosomes and many phage and viral DNA molecules are circlular and comprise single replicon. E. coli genome begin bidirectional replication from the unique OriC .There is a single termination site roughly 180o opposite the only origin.,The long, linear DNA molecules of eukaryotic chromosomes consist of multiple replicons, each with its own origin. When replication forks from adjacent replication bubbles meet, they fuse to form the completely replicated DNA. No distinct termini are required.,3.2.1 form,Carins,E.coli DNA,3.2.2 Rolling circle form,3.2.3 D Loop form,3.3.1 Enzymes and proteins in pro DNA replication,3.3.1.1 DNA polymerases（DNA pols） There are at least 5 kinds of DNA pols in E.coli; Replication- DNA pol III Delete primer- DNA pol I Repair- DNA pol II;IV;V,3.3 Enzymes and proteins needed in DNA replication,表3-1 大肠杆菌DNA聚合酶的比较,5 3 exonuclease activity：removing RNA primer 35 exonuclease activity: is used to excise bases that have been added to DNA incorrectly proofreading； Klenow fragment: The larger cleavage product (68 kD) of DNA polby proteolytic treatment (Bacillus subtilis protease), lacking 53 exonuclease activity；,DNA polymerase I,Nick translation: DNA polymerase I has the ability to start replication in vitro at a nick in DNA. At a point where a phosphodiester bond has been broken in a double-stranded DNA, the enzyme extends the 3-OH end. As the new segment of DNA is synthesized, it displaces the existing homologous strand in the duplex. The major technique for introducing radioactively labeled nucleotides into DNA in vitro,DNA pol III holoenzyme,The E.coli DNA polymerase III holoenzyme is a 900 kD complex that contains 10 proteins and organized into four types of subcomplex； There are two copies of the catalytic core. Each catalytic core contains a subunit (the DNA polymerase activity), a subunit (35 exonuclease), and a subunit (stimulates exonuclease)；,