DNA Replication Lecture 3 PDF

DNA REPLICATION by Dr.Ezat Mersal Replication Models of Replication DNA Topoisomerase Replication Fork Differences between DNA Polymerase I, II and III How is DNA Synthesized?  Replication : is the process by which DNA makes a copy of itself during cell di...

DNA REPLICATION by Dr.Ezat Mersal Replication Models of Replication DNA Topoisomerase Replication Fork Differences between DNA Polymerase I, II and III How is DNA Synthesized?  Replication : is the process by which DNA makes a copy of itself during cell division. DNA has to be copied before a cell divides DNAis copied during the S or synthesis phase of interphase New cells will need identical DNA strands © 2015 John Wiley & Sons, Inc. All rights reserved. © 2015 John Wiley & Sons, Inc. All rights reserved.  There 8 are 3 models of replication : 0 1. Conservative: the parental molecule directs synthesis of an entirely new double-stranded molecule, such that after one round of replication, one molecule is conserved as two old strands. 2. Semi Conservative: the two parental strands separate and each makes a copy of itself. After one round of replication, the two daughter molecules each comprise one old and one new strand. 3. Dispersive: material in the two parental strands is distributed randomly between two daughter molecules. © 2015 John Wiley & Sons, Inc. All rights reserved. © 2015 John Wiley & Sons, Inc. All rights reserved.  Daughter DNA is a double helix with 1 parent strand and 1 new strand. 1 strand serves as the template for new strand © 2015 John Wiley & Sons, Inc. All rights reserved.  Each strand of the parent DNA is used as a template to make the new daughter strand DNA replication makes 2 new complete double helices each with 1 old and 1 new strand © 2015 John Wiley & Sons, Inc. All rights reserved.  Site where replication begins is called ori 1 in E. coli ( Ori C) 1,000s in human (ori) Strands are separated to allow replication machinery contact with the DNA Many A-T base pairs Note anti-parallel chains © 2015 John Wiley & Sons, Inc. All rights reserved.  Prokaryotes (bacteria) have a single bubble Eukaryotic chromosomes have MANY bubbles © 2015 John Wiley & Sons, Inc. All rights reserved. 1. Unzip the double helix structure of the DNA molecule. Thisis carried out by an enzyme called helicase which breaks the weak hydrogen bonds holding the complementary bases of DNA together (A with T, C with G). Theseparation of the two single strands of DNA creates a “Y” shape called a Replication Fork. The two separated strands will acts as templates for making the new strands of DNA. © 2015 John Wiley & Sons, Inc. All rights reserved.  DNA helicase © 2015 John Wiley & Sons, Inc. All rights reserved. Single-Strand Binding Proteins SSBs attach and keep the 2 DNA strands separated and untwisted DNA HELICASE + SSBs leads to DNA unwinding and active replication Enzyme Topoisomerase attaches to the 2 forks of the bubble to relieve stress on the DNA molecule as it separates © 2015 John Wiley & Sons, Inc. All rights reserved. © 2015 John Wiley & Sons, Inc. All rights reserved. © 2015 John Wiley & Sons, Inc. All rights reserved.  Bidirectional movement of the DNA replication machinery © 2015 John Wiley & Sons, Inc. All rights reserved. 2. One of the strands is oriented in the 3’ to 5’ direction (towards the replication fork), this is the leading strand. The other strand is oriented in 5’ to 3’ direction (away from the replication fork) this is the lagging strand. As a result of their different orientations, the two strands are replicated differently. © 2015 John Wiley & Sons, Inc. All rights reserved.  In prokaryotes, there are 3 types of polymerase (I, II, III). An enzyme (Pol III) catalyzes the addition of a nucleotide to the growing DNA chain, (processing) Nucleotide enters as a nucleotide tri-PO4 Bidirectional synthesis of the DNA double helix © 2015 John Wiley & Sons, Inc. All rights reserved. © 2015 John Wiley & Sons, Inc. All rights reserved. © 2015 John Wiley & Sons, Inc. All rights reserved. Segment © 2015 John Wiley & Sons, Inc. All rights reserved.  DNA polymerase can only ADD nucleotides to a growing polymer Another enzyme, primase, synthesizes a short RNA chain called a primer DNA/RNA hybrid for this short stretch Base pairing rules followed (BUT A-U) Later removed, replaced by DNA and the backbone is sealed (ligated) In eucaryotic cells, DNA polymerase Alpha is responsible for the synthesis of primers. © 2015 John Wiley & Sons, Inc. All rights reserved.  Simple addition of primer along leading strand Many primers are needed along the lagging strand © 2015 John Wiley & Sons, Inc. All rights reserved.  Along the leading strand ‘ DNA polymerase reads 3’ 5’ from the RNA primer ‘ Synthesis proceeds 5’  3’ with respect to the new daughter strand The nucleotides are usually added 5’  3’ © 2015 John Wiley & Sons, Inc. All rights reserved.  Along the lagging strand Other daughter strand (lagging) is also synthesized 5’3’ because that is only way that DNA can be assembled However the template is also being read 5’3’ ‘ Compensate for this by feeding the DNA strand through the polymerase, and many primers and make many short segments that are later joined (ligated) together (= Okazaki fragments) so it called the lagging strand © 2015 John Wiley & Sons, Inc. All rights reserved.  This type of replication is called discontinuous as the Okazaki fragments will need to be joined up later.  Once all of the bases are matched up (A with T, C with G), an enzyme called Exonuclease strips away the primer(s). The gabs were then filled by yet more complementary nucleotides.  Finally, an enzyme called DNA Ligase seals up the sequence of DNA into two continuous double strands. © 2015 John Wiley & Sons, Inc. All rights reserved. © 2015 John Wiley & Sons, Inc. All rights reserved. © 2015 John Wiley & Sons, Inc. All rights reserved.  Basic genetics : a human approach / BSCS. Dubuque, IA, Kendall/Hunt Pub. Co., c1999. 147 p. QH431.B305 1999 Genes, ethnicity, and ageing. Edited by Lincoln H. Schmitt, Leonard Freedman, Rayma Pervan. Nedlands, Australia, Centre for Human Biology, University of Western Australia ; Singapore, River Edge, NJ, World Scientific, c1995. 100 p.QH455.G45 1995 Genetic polymorphisms and susceptibility to disease. Edited by M. S. Miller and M. T. Cronin. New York, Taylor & Francis, 2000. 266 p. Ogawa, T., & Okazaki, T. (1980). Discontinuous DNA replication. Annual review of biochemistry, 49(1), 421-457. Kunkel, T. A., & Bebenek, K. (2000). DNA replication fidelity. Annual review of biochemistry, 69(1), 497-529. Benkovic, S. J., Valentine, A. M., & Salinas, F. (2001). Replisome- mediated DNA replication. Annual review of biochemistry, 70(1), 181-208. Ogawa, T., & Okazaki, T. (1980). Discontinuous DNA replication. Annual review of biochemistry, 49(1), 421-457. Kunkel, T. A., & Bebenek, K. (2000). DNA replication fidelity. Annual review of biochemistry, 69(1), 497-529. Benkovic, S. J., Valentine, A. M., & Salinas, F. (2001). Replisome- mediated DNA replication. Annual review of biochemistry, 70(1), 181-208.