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1、Chapter 04: DNA Replication,Watson & Crick：一种遗传物质，必须能行使两种功能，即自我复制和对细胞的高度特异性的影响。,遗传物质的基本属性：自我复制控制性状的表达产生突变,生物体的遗传信息以密码的形式编码在DNA分子上，表现为特定的核苷酸排列顺序； 遗传信息通过DNA的复制由亲代传递给子代； DNA双螺旋模型的提出是DNA复制的理论基础。,DNA是生物遗传的主要物质基础：,复制的复杂性,DNA复制起始控制机理知之甚少,1 overview of DNA replication,1.1 semi-conservative replication Defin
2、ition: during replicaiton, the two parental strands separate and each acts as a template to direct the synthesis of a new complementary daughter strand following the normal base-pairing rules. 1958 Meselson & Stahl E.coli Radioactive isotope(15N) labelCsCl density gradient centrifugation,15N,15N/14N
3、,14N 15N/14N,First replicaiton,second replicaiton,parental strands,意义：保证DNA代谢的稳定性。经过多代的复制，子代DNA仍可以保持与最初亲本的一致性。 稳定性是相对的，变异是绝对的,1.2 replicaiton unit and direction,1.2.1 Replicon A unit of the genome in which DNA contain a region from origin to terminus. Origin is the DNA sequence where a replicon init
4、iates its replication. Origins tend to be AT-rich to make opening easier Terminus is the DNA sequence where a replicon usually stops its replication,All prokaryotic chromosomes and many bacteriophage and viral DNA molecules are circlular and comprise single replicons.There is a single termination si
5、te roughly 180o opposite the unique origin. In all the cases, the origin is a complex region where the initiation of DNA replication and the control of the growth cycle of the organism are regulated and coordinated.,The long, linear DNA molecules of eukaryotic chromosomes consist of mutiple regions,
6、 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,1.2.2 directionUnidirectionalBidirectional(more common),1.2.3 methods for mapping direction and origins,1.2.3.1 replication dire
7、ction autoradiography E.coli steps： E.coli先用高放射性的3H-dTTP标记； 几分钟后，E.coli转移入低放射性的3H-dTTP中继续标记； 提取DNA进行放射自显影； 根据底片感光银粒密度的分布判断复制的方向。 结论： 单向复制 银粒的密度：一端高，一端低； 双 中间高，两端低。,根据：越靠近origin 的基因转导的频率越高,4a 4b 3c 3d 2e 2f 1g 1h,复制的不同步性、断裂的随机性,replication origin,1.2.3.2 origins,分子杂交确定各基因片段的频率,1.3 primer,A common fea
8、ture of all DNA polymerases is that they cannot initiate synthesis of a chain of DNA de novo. All DNA polymerases require a 3OH priming end to initiate DNA synthesis. For DNA replication, a special RNA polymerase called a primase synthesizes an RNA chain that provides the priming end. A primer is a
9、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,ssDNA virus,RF(replicating form),No M13 RF,Rifampin,M13,Rifampin,M13 RF,M13 RF,M13,Rifampin,M13,expression, packaging, release,It
10、s necessary of RNA polymerase to synthesize a RNA in the formation of M13 RF；,after the formation of M13 RF, the inhibition of rifampin is invalid,Conclusion,The first evidence supporting RNA priming,Rifampin is an inhibitor of E.coli RNA polymerase,OK !,How ?,1.4 semi-discontinuous replication,Repl
11、ication fork,1968 Okazaki radioactivity label (3H-dTTP) + CsCl density gradient centrifugation label enters newly synthesized DNA in the form of short fragments(1000-2000 base in length) DNA ligase mutant strain which is sensitive to temperature in the temperature that ligase have no effection a lar
12、ge 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-s
13、tranded 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, each 53 ; then they are joined together to create an intact lagging strand. Semi-discontinuous replication-the lagging strand is
14、synthesized discontinuously and the leading strand is synthesized continuously,2 Prokaryotic DNA replication,2.1 enzymes and proteins for pro replication 2.1.1 DNA polymerases（DNA pols）,3 5 exonuclease activity: is used to excise bases that have been added to DNA incorrectly-proofreading； Klenow fra
15、gment: The larger cleavage product (68 kD)of DNA polby proteolytic treatment(Bacillus subtilis protease), lacking 53 exonuclease activity ；,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 3OH end. As the new segment of DNA is synthesized, it displaces the existing homologous strand in the duplex. a major technique for introducing radioactively labeled nucleotides into DNA in vitro,
