1. Online Counseling Resource YCMOU ELearning Drive… School of Architecture, Science and Technology Yashwantrao Chavan Maharashtra Open University, Nashik – 422222, India

2. OC-SBT044-U01-01 Introduction Programmes and Courses  SEP – SBT044 – CP01  SEP – SBI044 – CP01  SEP – SGS044 – CP01

3. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.3 Credits  Academic Inputs by Mrs. Rasika Bhore  M.sc (Microbiology) rasika.bhore@gmail.com

4. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.4 How to Use This Resource  Counselor at each study center should use this presentation to deliver lecture of 40-60 minutes during Face-To-Face counseling.  Discussion about students difficulties or tutorial with assignments should follow the lecture for about 40-60 minutes.  Handouts (with 6 slides on each A4 size page) of this presentation should be provided to each student.  Each student should discuss on the discussion forum all the terms which could not be understood. This will improve his writing skills and enhance knowledge level about topics, which shall be immensely useful for end exam.  Appear several times, for all the Self-Tests, available for this course.  Student can use handouts for last minutes preparation just before end exam.

5. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.5 Learning Objectives  After studying this module, you should be able to: Discuss the role of all enzymes participated in Replication.

6. School of Science and Technology, Online Counseling Resource… Introduction  Like all metabolic processes, DNA replication is also the combine action of many enzymes.  Physical, chemical, biochemical and mutation studies indicate that DNA replication is under the strict control of enzymes.  All these replication enzymes & proteins performs a specific task.  These entire complex of enzymes works together for replication & known as ‘DNA replicase system or replisome’. © 2007, YCMOU. All Rights Reserved.6

7. School of Science and Technology, Online Counseling Resource… Enzymes Of Replication  Following are the main enzymes of DNA replication. 1.Helicases 2.DNA gyrase or topoisomerase 3.Primases 4.DNA polymerases 5.DNA ligases © 2007, YCMOU. All Rights Reserved.7

8. School of Science and Technology, Online Counseling Resource… Helicases  Helicases mainly functions in separating the two annealed nucleic acid strands (DNA, RNA).  For the replication, it is necessary to separate the double stranded DNA strand which is done by Helicases.  For this activity they derived energy from ATP or GTP hydrolysis which is the output of breaking of hydrogen bonds between nucleotides during separation. © 2007, YCMOU. All Rights Reserved.8

9. School of Science and Technology, Online Counseling Resource… DNA Gyrase Or Topoisomerase  DNA gyrase is type II topoisomerase.  This enzyme relieves the topological stress of helical DNA structure created on strand separation.  The stress is removed by introducing the negative supercoils on other words by relaxing positive supercoils. © 2007, YCMOU. All Rights Reserved.9

10. School of Science and Technology, Online Counseling Resource… Primases  For the activity of DNA polymerase primers must be present on template.  DNA polymerase started to synthesize new DNA by adding nucleotides to primer.  These primers are the short segments necessary for synthesis of lagging strand in eukaryotes are produced by the enzyme Primase.  In bacteria, primase binds to the DNA helicase forming a complex called the primosome.  Primase is activated by DNA helicase where it then synthesizes a short RNA primer approximately 11 to 12 nucleotides long, to which new nucleotides can be added by DNA polymerase. © 2007, YCMOU. All Rights Reserved.10

11. School of Science and Technology, Online Counseling Resource… DNA Ligases  Lagging strand synthesis carried out by formation of okazaki fragments by polymerase III which adds nucleotides to 3’OH group of RNA primer.  Polymerase I then replaces the RNA fragments with DNA & fill the gaps.  Finally DNA ligase fill the nicks & finishes the synthesis of DNA.  It also functions in DNA repair. © 2007, YCMOU. All Rights Reserved.11

12. School of Science and Technology, Online Counseling Resource… Action Of Ligase In DNA Synthesis  The mechanism of DNA ligase is to form covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide with the 5' phosphate end of another.  ATP is required for the ligase reaction. © 2007, YCMOU. All Rights Reserved.12

13. School of Science and Technology, Online Counseling Resource… DNA Polymerase  DNA polymerase is the enzyme which synthesizes DNA.  It catalyzes the polymerization of deoxyribonucleotides along a DNA strand.  The newly-polymerized molecule is complementary to the template strand and identical to the template's partner strand.  DNA polymerase requires a magnesium ion as a co- factor to function properly.  No known DNA polymerase is able to begin a new chain (de novo), it can add a nucleotide onto only a preexisting 3'-OH group, and, therefore, needs a primer at which it can add the first nucleotide.  Thus the elongation of chain takes place. © 2007, YCMOU. All Rights Reserved.13

14. School of Science and Technology, Online Counseling Resource… Prokaryotic DNA Polymerases  E.coli have 5 known DNA polymerases: 1.Pol I: implicated in DNA repair; has both 5'- >3'(Polymerase) activity and 3'->5' (Proofreading) exonuclease activity. 2.Pol II: involved in replication of damaged DNA; has 3'->5' exonuclease activity. 3.Pol III: the main polymerase in bacteria (elongates in DNA replication); has 3'->5' exonuclease proofreading ability. 4.Pol IV: a Y-family DNA polymerase. 5.Pol V: a Y-family DNA polymerase; participates in bypassing DNA damage. © 2007, YCMOU. All Rights Reserved.14

15. School of Science and Technology, Online Counseling Resource… Proofreading Or Exonuclease Activity  DNA polymerases shows the 3’->5’ exonuclease activity.  Exonuclease activity double checks each nucleotide after it added.  This activity permits the enzyme to remove a newly added nucleotide & is highly specific for mismatched base pairs.  If the polymerase has added the wrong nucleotide, translocation of the enzyme to the position where the next nucleotide is to be added is halted.  This pause provides the opportunity for a correction.  Thus the 3’->5’ exonuclease activity removes the mispaired nucleotide, & he polymerase moves ahead.  This activity is known as ‘proofreading’. © 2007, YCMOU. All Rights Reserved.15

16. School of Science and Technology, Online Counseling Resource… DNA Polymerase I  Polymerase I is not a primary enzyme of replication, instead it performs a clean-up functions during replication, repair & recombination.  It is a single-chain protein with a mass of about 109,000 Da.  Each of its three enzymatic activities are encapsulated into distinct domains of the holoenzyme.  The proteolytic deletions can separate the 3’->5’ (proofreading) exonuclease domain.  The remaining fragment is large fragment or called Klenow fragment retains the polymerization & proofreading activities & widely used in recombinant DNA work. © 2007, YCMOU. All Rights Reserved.16

17. School of Science and Technology, Online Counseling Resource… DNA Polymerase III  Polymerase III is the primary enzyme complex having 10 types of subunits.  It was discovered by Thomas Kornberg and Malcolm Gefter in 1970.  The complex has high processivity and, specifically referring to the replication of the E.coli genome, works in conjunction with four other DNA polymerases (Pol I, Pol II, Pol IV, and Pol V).  DNA Pol III holoenzyme also has proofreading capabilities that correct replication mistakes by means of exonuclease activity working 3'->5'.  DNA Pol III is a component of the replisome, which is located at the replication fork. © 2007, YCMOU. All Rights Reserved.17

18. School of Science and Technology, Online Counseling Resource… Subunits Of DNA Pol III © 2007, YCMOU. All Rights Reserved.18 SUBUNITNO.OF SUBUNIT PER HOLOENZYME FUNCTION OF SUBUNIT α2 POLYMERIZATION ACTIVITY ε2 3’->5’ PROOFREADING EXONUCLEASE θ2 _ τ2 STABLE TEMPLATE BINDING √1 CLAMP LOADER ♪ 1 CLAMP OPENER ♪’♪’1 CLAMP LOADER ҳ 1 INTERACTION WITH SSB Ψ1 INTERACTION WITH √ & ҳ β4 DNA CLAMP REQUIRED FOR OPTIMAL PROCESSIVITY

19. School of Science and Technology, Online Counseling Resource… Structure Of DNA Pol III © 2007, YCMOU. All Rights Reserved.19

20. School of Science and Technology, Online Counseling Resource… Eukaryotic DNA Polymerases -1  Eukaryotes have at least 15 DNA Polymerases: 1.Pol α : acts as a primase (synthesizing a RNA primer). 2.Pol β: Implicated in repairing DNA, in base excision repair and gap-filling synthesis. 3.Pol γ: Replicates mitochondrial DNA. 4.Pol δ: Highly processive and has proofreading 3'->5' exonuclease activity. Thought to be the main polymerase involved in lagging strand synthesis. 5.Pol ε: Also highly processive and has proofreading 3'->5' exonuclease activity. Highly related to pol δ, and thought to be the main polymerase involved in leading strand synthesis. © 2007, YCMOU. All Rights Reserved.20

21. School of Science and Technology, Online Counseling Resource… Eukaryotic DNA Polymerases -2 6. η, ι, κ, and Rev1 are Y-family DNA polymerases and Pol ζ is a B-family DNA polymerase. These polymerases are involved in the bypass of DNA damage. 7. There are also other eukaryotic polymerases known, which are not as well characterized: θ, λ, φ, σ, and μ. 8. None of the eukaryotic polymerases have 5'->3' exonuclease activity; that function is carried out by other enzymes. © 2007, YCMOU. All Rights Reserved.21

22. School of Science and Technology, Online Counseling Resource… What We Learn………….  Helicase enzyme initiate the replication by separating the DNA strands.  The topological stress produced on separation is relieved by gyrase by introducing negative supercoils in DNA.  Primase synthesizes RNA primer without which DNA polymerase can’t elongate the chain of nucleotides.  There are 5 DNA polymerases in prokaryotes while 15 in eukaryotes.  Prokaryotic pol III have high processivity & have 3’->5’ exonuclease activity.  Prokaryotic pol I is primary enzyme of replication.

23. School of Science and Technology, Online Counseling Resource… Critical Thinking Questions  What will be the consequences if 3’->5’ proofreading activity has lost from DNA polymerase? © 2007, YCMOU. All Rights Reserved.23

24. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved. Tips For Critical Thinking Questions  Proofreading activity replaces the mismatched nucleotide with correct ones, thus, helps in accurately copying DNA.

25. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.25 Study Tips  Article Title: Enzymes & reactions at eukaryotic DNA replication fork. Author: Robert Bambara, R. Murante & L. Henricksen Publication: Vol. 272, No.8, Issue of Feb 21, 1997  Book Title: DNA Replication Author: Adam, Roger L. P

26. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.26 Study Tips www.en.wikipedia.org Polymerase, primase, Helicase etc. www.biochemistry.org DNA replication.

27. School of Science and Technology, Online Counseling Resource… Acknowledgement  www.vivo.colostate www.vivo.colostate  www.nature.com www.nature.com  www.medpham.yc.ac www.medpham.yc.ac  www.library.thinkquest.org www.library.thinkquest.org © 2007, YCMOU. All Rights Reserved.27

28. End of the Presentation Thank You !