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

2. OC-SBT053-CP1-01 Introduction Programmes and Courses  SEP – SBT053 – Unit 01

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 about the different types of an enzyme DNA Polymerase. Describe the functions of DNA Polymerase. Define ‘Primer’.

6. School of Science and Technology, Online Counseling Resource… Introduction  The search for an enzyme that could synthesize DNA began in 1955.  Work by Arthur Kornberg & colleagues led to the purification & characterization of DNA polymerase from E.coli cells.  That first enzyme was DNA polymerase I, which is a single polypeptide.  Later investigators found that E.coli contains atleast four other distinct DNA polymerases.

7. School of Science and Technology, Online Counseling Resource… DNA Polymerase………  DNA polymerase helps in replication of DNA.  It initiates replication by binding to a piece of single-stranded DNA.  It reads the template strand & form a complementary strand, which is identical to the partner of template strand.  The elongation of chain is carried out by adding deoxyribonucleotides to DNA.

8. School of Science and Technology, Online Counseling Resource… Prokaryotic DNA Polymerase  Bacteria (E.coli) have 5 known DNA polymerases: Pol I: implicated in DNA repair; has both 5'->3'(Nick translation) and 3'->5' (Proofreading) exonuclease activity. Pol II: involved in replication of damaged DNA; has 3'->5' exonuclease activity. Pol III: the principal replication enzyme. It has 3'->5' exonuclease proofreading ability. Pol IV: a Y-family DNA polymerase. Pol V: a Y-family DNA polymerase; participates in bypassing DNA damage.

9. School of Science and Technology, Online Counseling Resource… Eukaryotic DNA Polymerase  Eukaryotes have at least 15 DNA Polymerases: Pol α : acts as a primase (synthesizing a RNA primer), and then as a DNA Pol elongating that primer with DNA nucleotides. Pol β: implicated in repairing DNA. Pol γ: replicates mitochondrial DNA. Pol δ: main polymerase on the lagging strand, it is highly processive and has 3'->5' exonuclease activity. Pol ε: primary leading strand DNA polymerase, and also highly processive and has 3'->5' exonuclease activity. η, ι, κ, and Rev1 are Y-family DNA polymerases and Pol ζ is a B-family DNA polymerase are involved in the bypass of DNA damage. There are also other eukaryotic polymerases known, which are not as well characterized: θ, λ, φ, σ, and μ. Only the polymerases that deal with the elongation (γ, δ and ε) have proofreading ability (3'->5' exonuclease).

10. School of Science and Technology, Online Counseling Resource… Structure Of DNA Polymerase  DNA polymerases have highly conserved structure, which means that their overall catalytic subunits vary from species to species.  Conserved structures usually indicate important, irreplicable functions of the cell, the maintenance of which provides evolutionary advantages.

11. School of Science and Technology, Online Counseling Resource… Function Of DNA Polymerase  DNA polymerase plays a vital role in polymerization.  Investigation on DNA polymerase I, have put forward the two central requirements of DNA polymerization: 1.DNA polymerase require a ‘Template’. 2.DNA polymerase require a ‘Primer’.

12. School of Science and Technology, Online Counseling Resource… ‘Template’ For Polymerization  Polymerization reaction of DNA polymerase is guided by a ‘template’ DNA strand.  According to the base-pairing rules predicted by Watson & Crick, where the guanine is present in template, a cytosine deoxynucleotide is added to the new strand & so on.  This discovery is important as it provided a chemical basis for accurate semiconservative DNA replication.  It also represented the first example of the use of a template to guide a biosynthetic reaction.

13. School of Science and Technology, Online Counseling Resource… ‘Primer’ Primer is a strand segment (complementary to the template) with free 3’-hydroxyl group to which a nucleotide can be added.

14. School of Science and Technology, Online Counseling Resource…  DNA polymerase can add free nucleotides to only the 3’ end of the newly-forming strand.  This results in elongation of the new strand in a 5'-3' direction.  Part of the new strand (primer) must already be in place, DNA polymerase can only add a nucleotide onto a preexisting strand.  Primers consist of RNA and DNA bases with the first two bases always being RNA, and are synthesized by enzyme called primase. ‘Primer’ For Polymerization

15. School of Science and Technology, Online Counseling Resource… Application of DNA Polymerase In Research  DNA polymerase I obtained from E. coli is used extensively for molecular biology research.  It’s 5' -> 3' exonuclease activity can be simply removed from the holoenzyme to leave a useful molecule called the Klenow fragment.  Klenow fragment of DNA polymerase I has only 3’ -> 5‘ exonuclease activity, it is widely used in molecular biology.

16. School of Science and Technology, Online Counseling Resource… What We Learn………….  DNA Polymerase is an enzyme mainly participates in DNA replication.  Eukaryotes contain 15 types of DNA polymerases while prokaryotes contain 5 types.  DNA polymerase is made up of different sub-units having very complex structure.  only γ, δ and ε have proofreading ability i.e. 3'->5' exonuclease activity.  For the polymerization carried out by DNA polymerase, template & primer is required.  Klenow fragment have research value.

17. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.17 Critical Thinking Questions  What is mean by proofreading activity? If it is absent in DNA polymerase then what will be the consequences?

18. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved. Tips For Critical Thinking Questions  Checking & removal of wrong bases added in replication. It may leads in mutation.

19. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.19 Study Tips  Book Title: Lehninger ‘Principles Of Biochemistry’ Author: David Nelson Michael Cox  Book Title: Genetics Author: Peter J. Russell www.en.wikipedia.org DNA polymerase

20. End of the Presentation Thank You