DNA metabolism. DNA Replication DNA Repair DNA Recombination

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1 DNA metabolism DNA Replication DNA Repair DNA Recombination Chutima Talabnin Ph.D. School of Biochemistry,Institute of Science, Suranaree University of Technology

Central Dogma or Flow of genetic information http://www.youtube.com/watch?

2 Central Dogma or Flow of genetic information Replication process (Mcgrahill)

3 DNA template DNA replication How to keep the genetic information to next generation??? DNA replication 1. DNA replication is Semiconservative Each DNA stand serves as a template for the synthesis of new strand, producing two new DNA molecule. New DNA molecule Old strand (DNA templete) + New strand (Complementary strand)

4 2. Replication begins at an Origin and usually proceeds bidirection Dose replication begin at random places or unique point? After initiation at any point on the DNA. Dose replication proceed in one direction or both?

3. DNA replication proceeds in a 5 ----> 3 direction A new stand of DNA is always synthesis in the 5 ---> 3 direction.

5 3. DNA replication proceeds in a > 3 direction A new stand of DNA is always synthesis in the 5 ---> 3 direction. Because the two strand of new molecules DNA are antiparallel, the strand serving as the template is read from its 3 end toward its 5 end. How can both template strands be synthesized simultaneously? One strand is synthesis continuously using 3-5 template strand and called leading strand. Another strand call Okazaki fragments or lagging strand which is synthesized discontinuously using 5-3 template strand

DNA is synthesized by DNA polymerase DNA polymerase I 1.

d(nmp) n+1 + PPi Lengthened DNA Step1 : DNA polymerase requires

synthesis of new strand by adding complementary dntp (datp, dgtp,

6 DNA is synthesized by DNA polymerase DNA polymerase I 1. Polymerase activity d(nmp)n + dntp (DNA primer) Mg 2+ DNA polymerase d(nmp) n+1 + PPi Lengthened DNA Step1 : DNA polymerase requires primer binding to DNA template Step 2: DNA polymerase catalyze the synthesis of new strand by adding complementary dntp (datp, dgtp, dctp, and dttp) at 3 OH of lasted nucleotide which connect in each other by phosphodiester bond

7 DNA is synthesized by DNA polymerase (cont.) > 5 Exonuclease activity Double check each nucleotide at 3 end of DNA strand after it is added Once mismatch base pair happen, the enzyme catalyze by removing a newly added nucleotide Proofreading activity to give accuracy of polymerization of new strand However, when base selection and proofreading are combined, DNA polymerase leaves behind one net error for every bases added. How to correct them?? > 3 Exonuclease activity Remove one or a few nucleotides at 5 end End step of replication : primer is removed away from new strand Excision repair activity : to correct mismatch base repair 3 --> 5 Exonuclease activity 5 --> 3 Exonuclease activity A

8 Properties of the DNA Polymerases of E. coli Property Pol I Pol II Pol III (core) Mass (kd) , 27.5, 8.6 5' --> 3' Polymerization activity Yes Yes Yes 3' -->5 Exonuclease activity (Proofreading activity) 5' --> 3' Exonuclease activity (Excision repair activity) Polymerization rate (nucleotide / sec) Yes Yes Yes Yes No No ,000 Reference : Adapted from Kornberg, A., and Baker, T. A., DNA Replication, 2nd ed.: New York: W. H. Freeman and Co. and Kelman, Z., and O Donnell, M., DNA polymerase I is catalyzed by Protease enzyme. It cleavage into small unit (5 3 exonuclease activity) large subunit or called Klenow fragment (polymearse activity and 3-5 exonuclease activity)

DNA replication in E. coli Initiation step, Elongation step, Termination step Replication requires not just a single DNA polymerase but 20 or more different enzymes and proteins.

9 DNA replication in E. coli Initiation step, Elongation step, Termination step Replication requires not just a single DNA polymerase but 20 or more different enzymes and proteins. This entire complex called DNA replicase system or Replisome 1. Initiation step Origin of replication; Ori Ori C contain two series of short repeats : Three repeats of a 13 bp sequence Four repeats of 9 bp sequence Dna A Dna B (Helicase) Single strand DNA binding protein (SSB)

2. Elongation step Helicase DNA Gyrase (DNA Topoisomerase II) Primase RNA primer (5 --3 ) consist of 10-60 Ribonucleotide DNA polymerase III DNA

10 2. Elongation step Helicase DNA Gyrase (DNA Topoisomerase II) Primase RNA primer (5 --3 ) consist of Ribonucleotide DNA polymerase III DNA polymerase I (5-3 exonuclease activity + polymerase activity) DNA ligase DNA Gyrase

12 Synthesis of Okazaki fragment

Coordination of leading and lagging strand synthesis Both strands are produced by a single asymmetric DNA polymerase III dimer The lagging strand is looped so that DNA synthesis

13 Coordination of leading and lagging strand synthesis Both strands are produced by a single asymmetric DNA polymerase III dimer The lagging strand is looped so that DNA synthesis proceeds steadily on both leading and lagging strand template at the same time Overall direction of Replication

14 3. Termination step Terminal region contain multiple copies of a 20 bp sequence called Ter and usually locate at opposite to Ori. The two replication fork of the E.coli chromosome meet at a terminal region. Ter sequence function as binding sites for a protein called Tus (terminal utillization substance). Ter-Tus complex can arrest a replication fork Two daughter DNA molecules are linked in each other called Catenanes.

15 Separation of the Catenanes circles in E. Coli requires topoisomerase IV

16 DNA replication in Eukaryotic cell Origin of replication called Autonomously replicating sequences (ARS) or Replicator. The rate of replication fork movement in eukaryotic cell is 50 nucleotide/sec Eukaryote chromosome contain million of nucleotide. How to finish in short time? DNA polymerase α = polymerase activity for RNA or DNA primer synthesis DNA polymerase = polymerase proofreading activity (DNA pol III in E.coli) DNA polymerase = polymerase proofreading exonuclease activity (DNA pol I in Ecoli)

How to start the replication Origin recognition complex (Orc : Orc1 to Orc6) bind to Autonomously replicating sequences (ARS) Cdc6 and Cdt1 bind to Orc mediate the loading of Mcm2 to Mcm7 which act

17 How to start the replication Origin recognition complex (Orc : Orc1 to Orc6) bind to Autonomously replicating sequences (ARS) Cdc6 and Cdt1 bind to Orc mediate the loading of Mcm2 to Mcm7 which act as a helicase. Activation of DNA polymerase α --> ---> to synthesize both leading and Okasaki fragment David M. Gilbert Evaluating genome-scale approaches to eukaryotic DNA replication Nature Reviews Genetics 11, (October 2010) doi: /nrg2830

18 DNA replication in vitro = Polymerase chain reaction (PCR)

Maintaining the integrity of the information in DNA is a cellular essential, supported by a complex set of DNA repair system.

19 DNA Repair DNA can become damage by a variety of processes, some spontaneous, other catalyzed by environmental agent. Replication itself can very occasionally damage the information content in DNA when error introduce mismatched base pair (such as G pair with C) Maintaining the integrity of the information in DNA is a cellular essential, supported by a complex set of DNA repair system. Mutation in DNA mrna (contain mutation sequence) Protein with abnormal functions (Gain or Loss of function) Abnormal cell (Cancer) DNA Repair DNA without mutation mrna Protein with Normal functions Normal cell

20 Type of DNA repair system in E.Coli Mismatch repair Base excision repair Nucleotide excision repair Direct repair

21 Mismatch repair (During replication) The mismatches are always corrected to reflect the information in the old strand (template), so the repair system must somehow discriminate between the template and the newly synthesized strand. Tagging the template DNA with methyl group can distinguish old from newly synthesized strand. Strand discrimination is based on the action of Dam methylase which catalyze by adding methyl group at the N6 position of all adenine with 5 GATC sequence.

22 Mismatch repair (cont.) MutL protein form a complex with MutS protein, and the complex bind to all mismatch base pair MutH protein bind to MutL-MutS complex and encounter to nearly GATC sequence. DNA on both side of mismatch is threaded throught the MutL MutS complex, creating the loop. MutH has a site-specific endonuclease activity which catalyzed cleavage of the unmethylated strand on 5 side of G at GATC CTAG 5

23 Mismatch repair (cont.) Which direction to start repairing? It depend on location of the cleavage site relative to mismatch. DNA helicase II is a well-characterized Escherichia coli enzyme capable of unwinding duplex DNA and known to be involved in methyl-directed mismatch repair

24 Excision Repair Base excision repair DNA glycosylases recognize particularly common DNA lesions such as the product of cytosine and adenine deamination. It remove the affected base by cleaving the N-glycosyl bond. AP site (apurinic or apyrimidinic site) 5-3 exonuclease and polymerase activity C > T transition A > G transition

25 Nucleotide excision repair Excision Repair (cont.) The key enzyme for nucleotide excision repair is Excinuclease which catalyze two specific endonucleolytic cleavage with distance range bases (E. coli) and bases (human). It distinguish from standard endonuclease.

26 Direct repair Phototreactivation of Thymine dimer Pyrimidine dimer result from an ultraviolet light induced reaction. DNA photolyases use energy derived from absorbed light to reverse the damage.

27 DNA Recombination The rearrangement of genetic information within and among DNA molecules Homologous genetic recombination : involve genetic exchange between two DNA molecules the share an extended region of nearly identical sequence. Site specific recombination : the exchanges occur only a particular DNA sequence DNA transposition : involve a short segment of DNA with remarkable capacity to move from one location in a chromosome to another

diversity in a population Before prophase, each homologous set of chromatids is not align to each other well.

28 Homologous genetic recombination : can take place between any two DNA molecules that share sequence homology Function of homologous genetic recombination In meiosis (germ line cell) : to ensure accurate chromosome segregation : to enhances genetic diversity in a population Before prophase, each homologous set of chromatids is not align to each other well. Crossing over effectively links together all four homologous chromatids, linkage that is essential to the proper segregation of chromosome in first meiosis division

29 Function of homologous genetic recombination Replication : to serve in the repair of stalled replication fork

30 Site specific recombination : to cleavage at specific target DNA sequence by recombinase and ligate the strand to new strand. Function : DNA integration : Regulation of gene expression

31 DNA transposition : involve a short segment of DNA (transposable element or transposon) with remarkable capacity to move from one location in a chromosome to another Transposons are the simplest of molecular parasites, adapted to replication passively within the chromosome of host cells. Some transposons contain a gene that are useful to the host cell and also provide for transposition. Classes of transposons Insertion sequence (simple transposon) contain only sequence required for transposition and the genes for protein the promote transposition process. Complex transposons contian one or more genes in addition to those need for transposition. The extra may confer resistance to antibiotic and enhance the survival change in the host cells.

Gene for purple pigment A Simplified explanation for jumping genes and their effect on the Kernel Color of Indian Corn The reddish streaks on these corn grains are caused

This results in white streaks or mottling rather than a solid purple grain. The duration of a transposon in this "turned off" position affects the degree of mottling.

32 Gene for purple pigment A Simplified explanation for jumping genes and their effect on the Kernel Color of Indian Corn The reddish streaks on these corn grains are caused by transposons if the transposon moves to a position adjacent to a pigment-producing gene, the cells are unable to produce the purple pigment. This results in white streaks or mottling rather than a solid purple grain. The duration of a transposon in this "turned off" position affects the degree of mottling. If the pigmentation gene is turned off long enough by a transposon, the grain will be completely unpigmented. The reddish-purple patterns caused by transposons may be blotches, dots, irregular lines and streaks.

BCMB Chapters 34 & 35 DNA Replication and Repair

BCMB Chapters 34 & 35 DNA Replication and Repair BCMB 3100 - Chapters 34 & 35 DNA Replication and Repair Semi-conservative DNA replication DNA polymerase DNA replication Replication fork; Okazaki fragments Sanger method for DNA sequencing DNA repair