微生物遺傳與生物技術 (Microbial Genetics and Biotechnology)

1. 微生物遺傳與生物技術(Microbial Genetics and Biotechnology) 金門大學 食品科學系 何國傑 教授

2. Bacterial DNA structure and replication

3. 1. Composition: (1) Base: Purines: A (adenine), G (guanine); Pyrimidines: in DNA : C (cytosine), T (thymine) in RNA: C (cytosine), U (uracil) (2) nucleoside: A base attaches to the 1’ end of a pentose (a sugar ( DNA: deoxyribose; RNA: ribose) in DNA: deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine in RNA: adenosine, guanosine, cytidine, uridine (3) nucleotide: phosphate(s) attaches to 3’ end of the pentose + one phosphate group: in DNA: dNMP - deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxycytidine monophosphate (dCMP), deoxythymidine monophosphate (dTMP) I.Structures of nucleic acids (DNA and RNA)

4. I.Structures of nucleic acids (DNA and RNA) in RNA: NMP - adenosine monophosphate (AMP), guanosine monophosphate (GMP), cytidine monophosphate (CMP), uridine monophosphate (UMP) + two phosphate groups: dNDP; NDP (D: di-) dADP, dGDP, dCDP, dTDP; ADP, GDP, CDP, UDP + three phosphate groups: dNTP; NTP (T: tri-) dATP, dGTP, dCTP, dTTP; ATP, GTP, CTP, UTP

5. I.Structures of nucleic acids (DNA and RNA)

6. I.Structures of nucleic acids (DNA and RNA)

7. I.Structures of nucleic acids (DNA and RNA)

8. I.Structures of nucleic acids (DNA and RNA) Structure - Double helix（雙螺旋） a. H bond（氫鍵）: AT vs. GC b. Chargaff’s rule: in natural DNAs, the total amount of A is always equal to the total amount of T; The total amount of G is always equal to the total amount of C. c. Antiparallel（反向平行的）: 5’ to 3” direction d. Complementarity（互補性） e. Hydrophobic (趨水性) vs hydrophilic (親水性) f. covalent bond (共價鍵) vs non-covalent bomd (非共價鍵)

9. I.Structures of nucleic acids (DNA and RNA) 2. The double helix: In the early 1950s, the X-ray diffraction studies of Franklin and Wilkins showed that DNA is double helix. (1). The DNA chain: phosphodiester bonds join each deoxynucleotide- The phosphate attached to the 5’ position of the carbon of the deoxyribose of one nucleotide is attached to the hydroxyl group at the 3’ position of the carbon of the deoxyribose of the next nucleotide to form one strand of nucleotides, to form a 5’ to 3’, 5’ to 3’ backbone. (2). Base pairing: i. Chargaff rule: the concentration of G always equals the concentration of C and the concentration of A always equals the concentration of T no matter the source of the DNA.

10. I.Structures of nucleic acids (DNA and RNA) ii. Complementary base pair: Watson and Crick proposed that the two strands of the DNA are held together by specific hydrogen bonding between A and T, and G and C in opposite strands. iii. Antiparallel construction: The two chains of DNA run in opposite directions causing the 5’ phosphate end of one strand and the 3’ hydroxyl end of the other to be on the same end of double-stranded DNA molecule. iv. The major and minor grooves: Because the two strands of the DNA are wrapped around each other to form a double helix, the helix has two grooves between the two strands: major groove (the wide one) and minor groove (the narrow one).

11. I.Structures of nucleic acids (DNA and RNA)

12. I.Structures of nucleic acids (DNA and RNA) 5 end 3 end Each strand of the double helix is oriented in the opposite direction 3 end 5 end

13. I.Structures of nucleic acids (DNA and RNA)

14. I.Structures of nucleic acids (DNA and RNA) • High humidity (92%) DNA is called the B-form. It is probably close to the conformation of the most DNA in cells. • Lower humidity from cellular conditions to about 75% and DNA takes on the A-form • Plane of base pairs in A-form is no longer perpendicular to the helical axis • A-form seen when hybridize one DNA with one RNA strand in solution • A- and B-form are right-handed helix.

15. II. DNA replication A. Semiconservative（半保守性的）- 14N vs 15N B. template（模板）- leading strand vs lagging strand a. Okazaki fragments . D. Base pairing（鹼基配對） E. Enzymes（酶:酵素） a.substrates（受質）- dNTPs b.DNA primerase（引子合成） – RNA primer（引子） c.Helicase（解螺旋）- unwinding（解開） d.SSB (single strand binding proteins) e.DNA polymerase III（DNA聚合酶III）- Polymerization（聚 合作用）and proofreading（校正） f.Ligase（黏接酶） g.Topoisomerase II（拓樸酶II）

16. II. DNA replication • The basic process of replication involves polymerizing, or linking the precursors (nucleotides) into long chains or strands, using the sequence on the other strand as a guide. 2. DNA replication involves many enzymes. • The DNA polymerase attaches the first phosphate (called α) of one deoxyribonucleoside triphosphate to the OH of 3’ carbon of sugar of another deoxyribonucleoside triphosphate, and release the last two phosphates (called β and γ ) of the first deoxyribonucleoside triphosphate to produce energy for the reaction. The same reaction occurs over and over until a long chain is synthesized. • DNA polymerase can not start the synthesis of a new strand of DNA without a primer (a preexisting 3’ OH of a deoxyribonucleotide). • The short RNA is used as primer to initiate the synthesis of new DNA strand either by RNA polymerase or a special enzyme, primase. During DNA replication, special enzymes recognize and remove the RNA primer.

17. II. DNA replication 6. DNA polymerases also need a template strand to direct the nucleotide to be inserted at each step of polymerization reaction by complementary base-pairing. For example, the DNA polymerase will insert a T into the new strand when there is an A in the template strand. 7. Some proteins travel with DNA polymerase as part of a DNA replication complex, replicosome. The functions of these DNA polymerase accessory proteins are listed in table 1. 8. DNA replication is a semiconservative process: each of the new molecules will consist of one old conserved strand and one newly synthesized strand. (The Meselson-Stahl experiment). 9. Okazaki fragments and the replication fork (1) DNA polymerase can move only in the 3’- to 5’- direction on the template strand and synthesize the new DNA strand in the 5’- to 3’- direction.

18. II. DNA replication Base pairing provides the mechanism for replicating DNA

19. II. DNA replication

20. II. DNA replication Meselson-Stahl experiment

21. II. DNA replication

22. II. DNA replication (2) Because DNA molecule is an antiparallel structure, the DNA polymerase on one of the two strands would have to move in the wrong direction overall at the replication fork. (3) There are three types of DNA polymerase, I II, and III in E. coli. (4) On one template strand, DNA polymerase III initiates synthesis from an RNA primer and moves along the template DNA in the 3’-to 5’ direction. The newly synthesized DNA strand is referred as the leading strand. On the other strand, DNA polymerase also moves in the 3’-to 5’ direction, but works in opposition to the movement of the replication fork as a whole. In order to synthesize the second new strand (called lagging strand), DNA polymerase III makes short pieces of DNA fragments (called Okazaki fragments) from a new RNA primer about 10 to 12 nucleotides long synthesized by DnaG primase. (5) DnaG primase produces a new primer about once every 2 kilobases, recognizing the sequence 3’-GTC-5’. (6) DNA polymerase I removes the RNA primer by its 5’ exonuclease and replaces the RNA primer with DNA by its polymerization activity.

23. II. DNA replication (7) The Okazaki fragments are joined together by DNA ligase before the replication fork moves on. 10. Replication errors (1) To maintain the stability of a species, replication of DNA must be almost free of error. However, the wrong base is sometimes inserted into the growing DNA chain. Mismatches can occur when the bases take on forms called tautomers, which pair differently from the normal form of the base. (2) The cell can reduce mistakes during replication through one of the following ways: i. Editing function - Editing function can be performed by either DNA polymerase or other proteins. The 3’ exonuclease of DNA polymerase can remove the last incorrectly inserted base. The editing proteins probably recognize a mismatch because the mispairing will cause a minor distortion in the structure of double helix of the DNA. ii. Methyl-directed mismatch repair – The state of methylation of the DNA strands allows the mismatch repair system of E. coli to distinguish the new strand from the old strand after replication.

24. Tautomerization

25. II. DNA replication

26. II. DNA replication Looping models for replication in eukaryotes allow DNA polymerases to move in the same direction on the leading and lagging strands. In prokaryotic replication, the core DNA polymerase III is composed of two α-subunits. Additional proteins, PCNA and RF-C are involved in the eukaryotic replication complex.

27. II. DNA replication 11. Replication of bacterial chromosome and cell division (1) Most bacteria only have one circular chromosome that means there is only one unique DNA molecule per cell. In some bacteria, chromosome replicates but the cell for some reason does not divide. (2) The DNA replication initiates at a unique site called origin of chromosome replication (oriC). i. A primosome (consisting of DnaA, B, C and other proteins) may help the DnaG primase or other RNA polymerase to synthesize an RNA to start replication at oriC. (3) In E. coli, chromosome replication usually terminates in certain region but not at well-defined unique site. i. A termination region, ter, contains cluster of sites called ter sequences, which are only 22 bp long. ii. Cluster terA and terB bracker the termination region. iii. Replication fork can pass terA in the clockwise direction but not in counterclockwise direction. iv. Replication fork can pass terB in the counterclockwise direction but not in clockwise direction. v. It needs proteins help. In E. coli they are called terminus ultilization substance (Tus).

28. II. DNA replication

29. II. DNA replication

30. II. DNA replication 12. According to the Watson-Crick structure, the two strands are wrapped around each other about once every 10.5 base pairs to form the double helix. If two strands are wrapped around each other more than once every 10.5 base pairs, the DNA is said to be positively supercoiled, and to be negatively supercoiled if less than 10.5 base pairs. 13. The supercoiling of DNA in the cell is modulated by enzymes called topoisomerases which bind to DNA, break one or both of the strands and pass the DNA strands through the break before resealing it, and will either introduce or remove supercoils from DNA.

31. II. DNA replication 14. There are two types of topoisomerases: TopI cuts one strand and pass the other strand through the break before resealing and will change DNA one supercoil at a time. TopII cuts both strands and pass two other strands from somewhere else in the DNA through the break before resealing and will change DNA two supercoils at a time. The major bacterial TopI removes negative supercoils from DNA. Bacteria have more than one type of TopII. TopII can remove negative supercoils from DNA. The major bacterial TopII is called gyrase and can add supercoils. 15. Gyrase acts by first wrapping the DNA around itself and then cutting the two strands before passing another part of the DNA through the cuts, thereby introducing two negative supercoils. Adding negative supercoils increases the stress in the DNA and so requires energy, here is ATP.

32. II. DNA replication

33. III. Chromosome replication is coordinated with cell division 1. The bacterial nucleoid (1) The bacterial chromosome is not enclosed in the nuclear membrane as eukaryotic chromosomes. (2) DNA is about 1000 times longer than the bacterium itself and must be condensed to fit in the cell but also must be folded in such a way that it is available for its functions. (3) The area that condensed chromosome located is called nucleoid. (4) The nucleoid is composed of 30 to 50 loops of DNA emerging from a more condensed region, or core. Most of the DNA loops are twisted up on themselves, called supercoiling. Whether core attachment sites on DNA are unique or random is not clear. However, the repeated sequences called rep that are almost identical in all bacteria have been implicated as sites at which the DNA might attach to core.