生物化学课件：DNA Metabolism

1、DNA Metabolism,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 hyp

3、othsis 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 ne

4、w 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? Do

5、es 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 ori

6、gin,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,Continu

7、ous 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 by

9、 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 transla

11、tion,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 r

13、epeats 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 pla

14、sma 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 o

15、f 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,Mi

16、smatch 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 P

17、olymerase for both leading and lagging strand synthesis.,Summary,What is DNA Replication? DNA Replication is governed by a set of fundamental rules. DNA Replication is a semiconservative. Replication begins at an origin and usually proceeds bidirectionally. DNA synthesis proceeds in a 53 direction a

18、nd is semidiscontinuous.,Process of DNA replication Enzymes involved in DNA replication Process of DNA replication Replication is very accurate Replication in eukaryotic cells is more complex,2.DNA repair,Mismatch repair（错配修复） Base-excision repair（碱基切除修复） Nucleotide-excision repair （核苷酸切除修复） Direct

19、repair（直接修复） Recombination repair (重组修复) Error-prone repair or SOS response (易错修复和应急反应),Mutations,Mutaion: a permanent change in the nucleotide sequence of DNA (substitution mutation, insertion mutation, deletion mutation) Silent mutation: mutation affects nonessential DNA or it has a negligible eff

20、ect on the function of a gene.,Mutations are linked to cancer,In mammals there is a strong correlation between the accumulation of mutations and cancer Mutagenesis and carcinogenesis Of the compounds known to be carcinogenic, more than 90% are also found to be mutagenic,All cells have multiple DNA r

21、epair systems,The number and diversity of repair systems reflect both the importance of DNA repair to cell survival and the diverse sources of DNA damage.,Mismatch repair,How to distinguish parental (template) strands from newly synthesized strands. Methylation and mismatch repair,Model for the earl

22、y steps of methyl-directed mismatch repair,Completing methyl-directed mismatch repair,The detailed mechanism of DNA mismatch repair in eukaryotes has not been worked out. It may differ in other ways from methyl-directed system used by bacteria.,Base-excision repair,DNA glycosylases(糖基化酶 ) recognize

23、particularly common DNA lesions (such as the products of cytosine and adenine deamination) and remove the affected base by cleaving the N-glycosyl bond. This cleavage creates an apurinic or apyrimidinic site in the DNA, commonly referred to as an abasic or AP site,Base-excision repair,Nucleotide-exc

24、ision repair,DNA lesions that cause large distortions (扭曲, 变形) in the helical structure DNA generally are repaired by the nucleotide-excision system.,Nucleotide-excision repair,eighth,fifth,22nd,sixth,Direct repair,Best characterized example is direct photoreactivation of cyclobutane(环丁烷) pyrimidine

25、 dimers, a reaction promoted by DNA Photolyases(光修复酶),Recombination repair,How to repair? Double-strands breaks Double-strand cross-links Lesions in single-stranded DNA, the complementary strand is itself damaged or absent,Error-prone repair or SOS response,It involves specialized DNA polymerase (DN

26、A polymerase V or IV) that can replicate past many of the DNA lesions that would normally block replication.,3. DNA recombination,Homologous genetic recombination Site-specific recombination DNA transposition,Homologous genetic recombination,It (also called general recombination) involves genetic ex

27、change between any two DNA molecules(or segments of the same molecule) that share an extended region of nearly identical sequence.,Recombination during meiosis (减数分裂),Models for recombinational DNA repair of stalled replication forks,Homologous genetic recombination has multiple functions,It contrib

28、utes to the repair of several types of DNA damage. It provides, in eukaryotic cells, a transient physical link between chromatids(染色单体) that promotes the orderly segregation of chromosomes at the first meiotic cell division. It enhances the genetic diversity in a population.,Site-specific recombinat

29、ion,The exchanges occur only at a particular DNA sequence. Site-specific recombination system consists of an enzyme called a recombi-nase and a short (20 to 200bp), unique DNA sequence where the recombinase acts (the recombination site),The outcome of site-specific recombination depends on the location and orientation of the recombination sites,Integratio