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1、DNA M etabolism,Contents,DNA replication DNA repair DNA recombination,1.DNA replication,Fundamental rules for DNA Replication Process of DNA replication,Fundamental rules for DNA Replication,What is DNA Replication? The copying of parental DNA to form daughter DNA molecules with identical nucleotide
2、 sequences,Who acts as DNA replication template?,DNA acts as a template for the replication and transmission of genetic information,Which DNA strand acts as a template for the replication?,DNA半保留复制的实验证据,第一代,第二代,细菌,（含,15,N-DNA),普通DNA,普通DNA,重DNA,重DNA,普通培养基,普通培养基,细菌DNA双链,密度梯度离心,15,N-DNA,14,N-DNA,The hy
3、pothsis of semiconservative replication (半保留复制) was proposed by Waston and Crick in 1953 and proved by Meselson and Stahl in 1957.,What is DNA semiconservative Replication(半保留复制)?,Each DNA strand serves as a template for the synthesis of a new strand, producing two new DNA molecules, each with one n
4、ew strand and one old strand. (当细胞分裂 DNA进行复制时，双螺旋结构解开而成为单链，按碱基互补配对原则合成新的互补链。子代细胞中的DNA双链，其中一股单链完全重新合成，另一股单链是亲代链的完整保留。),DNA Replication is governed by a set of fundamental rules,DNA Replication is a semiconservative,Question?,Are the parental DNA strands completely unwound before each is replicated? D
5、oes replication begin at random places or at a unique point? After initiation at any point in the DNA, does replication proceed in one direction or both?,The parental DNA strands are simultaneously unwound and replicated,Bidirectional Replication,Replication initiates at a unique point, called an or
6、igin,Replication origin,Replication forks,DNA Replication is governed by a set of fundamental rules,DNA Replication is a semiconservative Replication begins at an origin and usually proceeds bidirectionally,How can both strands be synthesized simultaneously?,DNA synthesis is semidiscontinuous,Contin
7、uous or leading strand(领头链) Discontinuous or lagging strand(随从链) Okazaki fragment (冈崎片段): a few hundred to a few thousand nucleotides,DNA Replication is governed by a set of fundamental rules,DNA Replication is a semiconservative. Replication begins at an origin and usually proceeds bidirectionally.
8、 DNA synthesis proceeds in a 53 direction and is semidiscontinuous.,Process of DNA replication,Enzymes involved in DNA replication,Nucleases DNA polymerases Ligases,DNA is degraded by Nucleases,Nucleases (DNases or RNases) Exonucleases and Endonucleases Restriction endonucleases,DNA is synthesized b
9、y DNA polymerases,53 Template Primer with a free 3-hydroxy group With or without activity of exonucleases (53 or 35),E coli has at least five DNA polymeases,DNA polymerase I DNA polymerase II: repair DNA polymerase III: the principal replication enzyme DNA polymerase IV and V: involved in an ususual
10、 form of DNA repair( identified in 1999),DNA polymerase I,It is not the primary enzyme of replication It performs a host of clean-up functions during replication, recombination,and repair Large or Klenow fragment (large fragment cleaved by subtilisin 枯草杆菌蛋白酶) lacks 53 exonulease activity,Nick transl
11、ation,ligases,DNA replication requires many enzymes and protein factors,DNA replicase system or replisome Helicases Topoisomerases DNA binding proteins(stabilizing separated strands) Primases DNA polymerase DNA ligases,The synthesis of a DNA molecule can be divided three stages,Initiation Elongation
12、 Termination (distinguished both by the reactions taking place and by the enzymes required),Initiation,Recognizing the replication origin Unwinding the DNA bidirectionally Creating two potential replication forks,OriC, the replication origin(复制起点 ) of E. coli chromosome, 245bp,contains three tandem
13、repeats of a 13 bp and four repeats of a 9 bp.,Initiation is the only phase of DNA replication that is known to be regulated, but it is regulated such that replication occurs only once in each cell cycle. The timing of replication is affected by DNA methylation and interactions with the bacterial pl
14、asma membrane.,Elongation,Leading strand synthesis Lagging strand synthesis,Coordination of leading and lagging strand synthesis,Both strands are produced by a single asymmetric DNA polymerase III dimer. This is accomplished by looping the DNA of the lagging strand ,bringing together the two points
15、of polymerization.,复制过程中各酶和蛋白质因子的作用,Termination,The protein Tus (terminus utilization substance) bind to ter (terminus),DNA replication process,Replication is very accurate,The fidelity of DNA replication Hydrogen bonds Base pair geometry Proofreading (Mismatch repair),Replication is very accurate,M
16、ismatch DNA polymerase:10-4 to 10-5 After proofreading: 10-6 to 10-8 After mismatch repair: 10-9 to 10-10,Replication in eukaryotic cells is more complex,Essential features are the same in prokaryotes and eukaryotes Multiple origins in eukaryotic cells. Polymerase for the synthesis of short primers Polymerase for both leading and lagging strand synthesis.,Summary,What is DNA Replication? DNA Replication is gov
