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: Describe the sequence or events of replication. Describe how replication initiates, carry out & terminates.

6. School of Science and Technology, Online Counseling Resource… Introduction  DNA molecule synthesis takes place in 3 stages: 1.Initiation 2.Elongation 3.Termination.  Different enzymes participated during these events which catalyzes different chemical reactions.  These events of replication have studied in E. coli which are described here. © 2007, YCMOU. All Rights Reserved.6

7. School of Science and Technology, Online Counseling Resource… Initiation Of Replication  Replication of the E. coli chromosome is initiated at a unique site, ‘oriC’.  ‘oriC’ consist of 245 bp, in which the DNA sequence are present which are highly conserved among bacterial replication origin.  The key sequences are two series of short repeats, three repeats of 13bp sequences & four repeats of 9bp sequence. © 2007, YCMOU. All Rights Reserved.7

8. School of Science and Technology, Online Counseling Resource… Proteins Required In Initiation PROTEINFUNCTION DnaA proteinRecognizes ‘ori’ sequence; opens duplex at specific sites in origin. DnaB protein (helicase)Unwinds DNA DnaC proteinRequired for DnaB boinding at origin HUDNA binding histone like protein that stimulates initiation Primase (DnaG protein)Synthesizes RNA primers SSB- single stranded DNA binding protein Binds single stranded DNA RNA polymeraseFacilitates DnaA activity DNA gyrase (DNA topoisomerase II)Relieves torsional strain generated by DNA unwinding Dam methylaseMethylates 5’ GATC sequences at oriC © 2007, YCMOU. All Rights Reserved.8

9. School of Science and Technology, Online Counseling Resource… Replication Initiates………  DnaA protein, a 52-kD polypeptide, is the initiation factor, which recognizes and binds to 9bp repeats.  DnaA proteins then recognizes & successively denatures the A=T rich DNA region (consensus sequence = 5'- GATCTNTNTTNTT) of 13bp.  Above process requires ATP & HU which prevents nonspecific initiation at sites other than oriC.  DnaB protein is loaded to oriC by DnaC protein (29 kD) in the form of a hexameric (DnaB:DnaC:ATP) 6 complex, but DnaC protein does not enter the protein assemblage at oriC. © 2007, YCMOU. All Rights Reserved.9

10. School of Science and Technology, Online Counseling Resource… Pre-priming Complex  Addition of DnaB protein completes assembly of the pre-priming complex.  DnaB protein has helicase activity which unwinds the DNA bidirectionally & creates two potential replication forks.  Molecules of SSB binds to single stranded DNA, stabilizing the separated strands & preventing renaturation.  Gyrase relieves the topological stress produced by DnaB helicase. © 2007, YCMOU. All Rights Reserved.10

11. School of Science and Technology, Online Counseling Resource… Formation Of Primosome  The addition of primase to the pre-priming complex completes the formation of the primosome, a protein machine of about 700 kD that synthesizes the RNA primer essential to DNA synthesis.  The AT-rich tandem repeats of oriC resemble sequences with which RNA polymerase interacts.  The two primosomes form at oriC, one for each replication fork.  Once the primosome has primed leading strand synthesis, it remains associated with the lagging strand and periodically primes Okazaki fragment synthesis, as required. © 2007, YCMOU. All Rights Reserved.11

12. School of Science and Technology, Online Counseling Resource… Elongation  Elongation phase of replication includes two distinct steps: leading strand synthesis & lagging strand synthesis.  DnaB moves in the 5' -> 3' direction along the leading strand template, hydrolyzing 2 ATPs for each base pair that it separates.  Protein-protein interaction between a ‘t’ subunit of the DNA polymerase III holoenzyme and DnaB is essential for rapid replication fork progression.  PriA protein is, like DnaB, a helicase associated with the primosome, but moves 3' -> 5' along the lagging strand.  The binding of SSB protein to the single-stranded regions created behind these proteins prevents re-annealing.  Leading & lagging strands are synthesized by a single DNA pol III dimer, which is accomplished by looping the DNA of the lagging strand bringing together the two points of polymerization. © 2007, YCMOU. All Rights Reserved.12

13. School of Science and Technology, Online Counseling Resource… Leading Strand Elongation - 1  Leading strand synthesis is straight forward begins with the synthesis by primase (DnaG protein) of a short RNA primer at replication origin.  DNA pol III then binds to primer & adds deoxyribonucleotides, thus elongates the strand.  DNA pol III uses one set of its core subunits to synthesize the leading strand continuously, while the other set of core subunits cycles from one to next okazaki fragment synthesizes the lagging strand, by looping it. © 2007, YCMOU. All Rights Reserved.13

14. School of Science and Technology, Online Counseling Resource… Lagging Strand Elongation - 2  Lagging strand synthesizes through formation of okazaki fragments.  DnaB helicase unwinds the DNA at replication fork as it travels along the lagging strand template in the 5’->3’ direction.  DNA primase synthesizes a short RNA primer.  A new β- sliding clamp is loaded at the primer by DNA pol III clamp loading complex.  Replication halts after synthesis of okazaki fragment & core subunits of DNA pol III dissociate from their β- sliding clamp & associate with new clamp.  This initiates synthesis of new okazaki fragment.  Thus the entire complex carry out coordinated DNA synthesis.  Once an okazaki fragment has been completed, its RNA primer is removed & replaced with DNA by DNA pol I & the remaining nick is sealed by DNA ligase. © 2007, YCMOU. All Rights Reserved.14

15. School of Science and Technology, Online Counseling Resource… Overview Of Elongation © 2007, YCMOU. All Rights Reserved.15

16. School of Science and Technology, Online Counseling Resource… Termination  Two replication forks of the circular E.coli chromosome meet at a terminus region containing multiple copies of a 20bp sequence called ‘Ter’ sequences.  These Ter sequences act as terminators that halts replication.  Clusters of three or four Ter sequences are organized into two sets inversely oriented with respect to one another.  One set blocks the clockwise-moving replication fork, and its inverted counterpart blocks the counterclockwise-moving replication fork.  Ter sequences function as binding sites for a protein called ‘Tus’.  The ‘Tus-Ter’ complex can arrest replication fork from only one direction.  Tus protein is a contrahelicase, That is, Tus protein prevents the DNA duplex from unwinding by blocking progression of the replication fork and inhibiting the ATP-dependent DnaB helicase activity. © 2007, YCMOU. All Rights Reserved.16

17. School of Science and Technology, Online Counseling Resource… ‘Ter’ Sequence Is Not Essential(?)  Mutations in either the Ter locus or the gene encoding Tus protein do not grossly affect DNA replication, demonstrating that ‘Ter’ sequences are not essential.  But these sequences may prevent over replication by one replication fork in case when other is delayed by any obstacle like DNA damage.  Thus either replication forks when encounters ‘Tus-Ter’ complex, the other fork halts when it meets the first fork leaving the circular progeny chromosomes intertwined by 20 to 30 coils about each other, a so-called catenated state.  These catenanes are separated by topoisomerase IV & then segregate into daughter cells at the cell division. © 2007, YCMOU. All Rights Reserved.17

18. School of Science and Technology, Online Counseling Resource… What We Learn………….  Replication initiates at ‘oriC’ where consensus sequences are present.  The DnaA protein recognizes the 13bp repeats & starts denaturing DNA.  Pre-priming complex establishes which carry out initiation with the help of proteins SSB & DNA gyrase.  Leading strand & lagging strand elongation carry out by DNA pol III core subunits at origin by extending the RNA primers formed by Primase.  β-sliding clamp of DNA pol III recycles to elongate lagging strand from one okazaki fragment to other.  ‘Ter-Tus’ complex helps in termination of replication.  Catenanes are formed which separated & then located in daughter cells.

19. School of Science and Technology, Online Counseling Resource… Critical Thinking Questions  What is the importance of DNA Topoisomerase enzyme in replication? Does replication carry out in its absence? Why? © 2007, YCMOU. All Rights Reserved.19

20. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved. Tips For Critical Thinking Questions  Topoisomerase relieves the stress on unwinding DNA. Absence of it leads in over winding of DNA.

21. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.21 Study Tips  Book Title: DNA In Detail Author: J.D.Watson  Book Title: Microbial Genetics Author: Abou-Sabe, Morad

22. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.22 Study Tips www.en.wikipedia.org DNA replication www.web.virginia.edu DNA replication

23. End of the Presentation Thank You !