Paper No. : 04 Genetic Engineering and Recombinant DNA Technology Module : 09 DNA Replication in Eukaryotes Principal Investigator: Dr Vibha Dhawan, Distinguished Fellow and Sr. Director The Energy and Resouurces Institute (TERI), New Delhi Co-Principal Investigator: Prof S K Jain, Professor, of Medical Biochemistry Jamia Hamdard University, New Delhi Paper Coordinator: Dr Mohan Chandra Joshi, Assistant Professor, Jamia Millia Islamia, New Delhi Content Writer: Dr. Bhaswati Banerjee, Assistant Professor, Gautam Buddha University, Greater Noida, UP Content Reviwer: Dr Mohan Chandra Joshi, Assistant Professor, Jamia Millia Islamia, New Delhi Genetic Engineering and Recombinant DNA Technology Biotechnology DNA Replication in Eukaryotes Page 1 of 33 Description of Module Subject Name Biotechnology Paper Name Genetic Engineering and Recombinant DNA Technology Module Name/Title DNA Replication in Eukaryotes Module Id 09 Pre-requisites Knowledge of DNA replication in prokaryotes Objectives To understand clearly the steps and mechanism of DNA replication in eukaryotes Keywords DNA polymerase, De novo synthesis, Proof reading, Exonuclease, Telomerase Genetic Engineering and Recombinant DNA Technology Biotechnology DNA Replication in Eukaryotes Page 2 of 33 INDEX LEARNING OBJECTIVES ABOUT THE MODULE I. INTRODUCTION A. Organization of Eukaryotic Chromosome B. Eukaryotic Cell Cycle and Replication Point C. Checkpoints in Eukaryotic Cell cycle II. EUKARYOTIC GENOME REPLICATION MACHINERY A. Origin of Replication and Pre-replication Complex B. Primosome and Replisome Complexes C. Eukaryotic DNA Polymerase D. Telomere and Telomerase III. MECHANISM OF DNA REPLICATION IN EUKARYOTES A. General Model of DNA Replication B. Enzymes Involved In Eukaryotic DNA Replication C. Steps Involved In DNA Replication in Eukaryotes IV. FIDELITY OF DNA REPLICATION IN EUKARYOTES V. SUMMARY VI. REFERENCES Genetic Engineering and Recombinant DNA Technology Biotechnology DNA Replication in Eukaryotes Page 3 of 33 GENOME REPLICATION FIDELITY • Eukaryotic MACHINERY • Genral Model chromosome • Enzyme structure and • Origin and Pre-RC complexes • Polymerase Cell-cycle • Primosome & • Steps in DNA selectivity • Checkpoints in Replisome Replication • Proofreading Cell cycle • Polymerases and • Mismatch repair Teloerase OVERVIEW MECHANISM Genetic Engineering and Recombinant DNA Technology Biotechnology DNA Replication in Eukaryotes Page 4 of 33 LEARNING OBJECTIVES: ● Eukaryotic Cell cycle and Replication Point ● Components of Eukaryotic Genome and Replication Machinery ● Steps In DNA Replication ● Fidelity of DNA Replication ● Comparison Between Prokaryotes & Eukaryotes Replication ABOUT THE MODULE NA replication is a highly conserved cellular activity that is known to occur invariably in all proliferating cells. It plays a pivotal role in faithful duplication D and transmission of genetic information from one generation to the next. As such, the fundamental mechanism underlying DNA replication remains conserved across all life forms. But as the organism moves higher up the evolutionary tree, the mechanism only becomes more elaborate with involvement of multiple steps and larger macromolecular machineries mediating each step. The fundamentals of DNA replication has been discussed in detail in “Module 08: DNA replication in Prokaryotes”. In the present module, we focus mainly on the macromolecular events exclusive to the eukaryotic DNA replication. This module begins with an overview of packaging of eukaryotic genome and eukaryotic cell cycle where the point of DNA replication is highlighted. The subsequent segment elaborates upon the components of eukaryotic DNA replication machinery and the multiprotein macromolecular complexes involved therein. Thereafter the module provides a detailed account of various steps involved in eukaryotic DNA replication followed by a note on maintenance of fidelity of DNA Replication. To conclude, we compare the replication process in prokaryotes and eukaryotes and underscore the important features in both. Genetic Engineering and Recombinant DNA Technology Biotechnology DNA Replication in Eukaryotes Page 5 of 33 I. INTRODUCTION Genomes of both prokaryotes and eukaryotes are DNA genomes, i.e., their genomes are composed of DNA and DNA binding proteins. As such, the overall mechanism of DNA replication remains conserved in prokaryotes and eukaryotes. Nevertheless, there exist certain striking differences between the genome of prokaryotes and that of eukaryotes and also in the pre-replication events. Single most important feature which distinguishes eukaryotes from prokaryotes is the occurrence of nucleus and the fact that the eukaryotic genome remains enclosed within a clearly defined nuclear envelope. Besides there are several other distinguishing features, to name a few, the eukaryotic chromosomes are linear and mostly many fold larger than prokaryotic genome as the former contain substantial amount of introns or non- coding DNA (Smith & Szathmary 1997). The DNA replication machinery and the components involved in eukaryotic DNA replication are far more complicated and elaborate compared to prokaryotic DNA replication machinery and must gain physical access to the DNA templates for carrying out DNA replication and this would be influenced by the physical state and organization of eukaryotic DNA (DePamphilis & Bell 2010). Before studying the mechanism of DNA replication, let us first understand how the enormous amount of DNA content in eukaryotic genome is organized inside a nucleus which is barely 4-6 μ in size. A. Organization of Eukaryotic Chromosome Eukaryotic DNA remains neatly packed inside the nucleus during most part of the cell cycle. Since the size of eukaryotic genome is many folds greater than the size of the nucleus, the DNA is compacted through multiple layers of packaging and organization from the 2 nm DNA duplex to the 1400 nm metaphase chromosome (Fig. 1.1) (Smith & Szathmary 1997; Alberts et al. 2014). The packaging of DNA begins with winding of DNA around octameric histone cores forming the 11 nm beads-on-string like nucleosomes. The nucleosomes are further coiled into solenoid like 30 nm fiber or the thinnest form of chromatin fiber achieving a packing ratio of 40 (Staynov & Proykova 2008). The solenoid like chromatin fiber is drawn into loops Fig. 1.1: Organization of DNA and compaction of eukaryotic arranged in spirals around a central core genome Source: http://philschatz.com/biology- of nuclear matrix forming the 300 nm book/contents/m44486.html#fig-ch14_02_06 Genetic Engineering and Recombinant DNA Technology Biotechnology DNA Replication in Eukaryotes Page 6 of 33 “looped domain” which is further condensed to form rosette of chromatin loops achieving a packing ratio of ~1000 and dimension of 700 nm. The most condensed packaging of chromosomes is observed in the mitotic chromosome that achieves a dimension of 1400 nm and packing ratio ~ 10000 (Alberts et al. 2014; Lodish et al. 2012) (Anon n.d.; Therman 1986). B. Eukaryotic Cell Cycle and Replication Point Cell cycle is defined as the sequence of events occurring between two consecutive mitotic divisions. The eukaryotic cells pass through complex yet coordinated and tightly regulated cell cycle with distinct phases of cellular events, namely the Interphase and the Mitotic phase. Interphase is further divided into three phases (Lodish et al. 2012; Alberts et al. 2014), the G1, S and the G2 phase as follows (Fig. 1.2): ● G1 Phase: It is the longest phase in cell G Cells cycle during which the genes encoding 0 DNA replication enzymes and S phase CdkC components are activated o mRNAs and proteins are synthesized during G1, but there is M no DNA replication Early G ● S-Phase: Point of DNA Replication 1 o Typically lasts for 6-8 hours, by the G end of which the DNA content of 2 the cell is doubled. Late S- phase G o S-phase CdkC, i.e., Cdk2-cyclins 1 regulate assembly of pre-replication complex at Origin followed by DNA Fig. 1.2: Schematic representation of cell cycle in eukaryotes replication o RNA and protein synthesis continues but DNA replication is ensured only once during the cell cycle ● G2-Phase: short gap phase lasting for 2-4 hours during which the G2-Cdk, Cdk1 associates with Cyclins A & B followed by activation of mitotic Cdks. o There is no further DNA replication, but RNA and protein synthesis continues C. Checkpoints in Eukaryotic Cell cycle Four distinct checkpoints have been identified in eukaryotic cell cycle (Fig. 1.3): G1/S checkpoint: Cells ascertain whether to enter into the division cycle or to enter into the G0 stage. Cell cycle arrest for cancer cells at this checkpoint leads to apoptosis. Genetic Engineering and Recombinant DNA Technology Biotechnology DNA Replication in Eukaryotes Page 7 of 33 ● If a cell crosses the G1/S restriction point, it enters into a point of no return, and thereafter, the cell is committed to divide or die. ● G2/M checkpoint: The cell arranges and checks post replication chromosomes during G2/M progression. This is a major checkpoint to ascertain that DNA replication and chromosome segregation has successfully occurred. These checkpoints function in response to DNA damage in a CIP dependent manner and prevent entry into M phase until the damage is repaired. ● S/G2 checkpoint: This checkpoint involves recognition of unreplicated DNA and inhibition of MPF activation causing S-phase arrest till the entire DNA replication is complete ● M-phase checkpoint: This operates during early mitosis in response to improper assembly