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Paper No. : 04 Genetic Engineering and Recombinant DNA Technology

Module : 09 DNA Replication in

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

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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

Objectives To understand clearly the steps and mechanism of DNA replication in eukaryotes

Keywords DNA , De novo synthesis, Proof reading, ,

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INDEX LEARNING OBJECTIVES

ABOUT THE MODULE

I. INTRODUCTION

A. Organization of Eukaryotic

B. Eukaryotic Cell Cycle and Replication Point

C. Checkpoints in Eukaryotic Cell cycle

II. EUKARYOTIC GENOME REPLICATION MACHINERY

A. and Pre-replication Complex

B. Primosome and Complexes

C. Eukaryotic DNA Polymerase

D. and Telomerase

III. MECHANISM OF DNA REPLICATION IN EUKARYOTES

A. General Model of DNA Replication

B. Involved In Eukaryotic DNA Replication

C. Steps Involved In DNA Replication in Eukaryotes

IV. FIDELITY OF DNA REPLICATION IN EUKARYOTES

V. SUMMARY

VI. REFERENCES

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GENOME REPLICATION FIDELITY • Eukaryotic MACHINERY • Genral Model chromosome • structure and • Origin and Pre-RC complexes • Polymerase Cell-cycle • Primosome & • Steps in DNA selectivity • Checkpoints in Replisome Replication • Proofreading Cell cycle • and • Mismatch repair Teloerase OVERVIEW MECHANISM

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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.

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I. INTRODUCTION

Genomes of both prokaryotes and eukaryotes are DNA genomes, i.e., their genomes are composed of DNA and DNA binding . 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 are linear and mostly many fold larger than prokaryotic genome as the former contain substantial amount of 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 cores forming the 11 nm beads-on-string like . 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

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“looped ” 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 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 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 cells at this checkpoint leads to apoptosis.

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● 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 of mitotic spindles and leads to arrest in anaphase. It prevents the activation of APC and the initiation of anaphase till the mitotic spindle apparatus is completely assembled and all kinetochores are properly attached to the spindle fibers.

Fig. 1.3: Eukaryotic cell cycle and checkpoints Source: Molecular Cell Biology | 6th edition

II. EUKARYOTIC GENOME REPLICATION MACHINERY

The first ever DNA polymerase to be discovered was bacterial DNA pol I by Arthur Kornberg in 1956. Thereafter Kornberg and other researchers discovered the other components of bacterial DNA replication system. Thus, most of the initial information about mechanism of DNA replication was obtained from prokaryotic system. Studies on eukaryotic DNA replication began in the 1960’s but were fully described towards the middle of 1980’s after the successful demonstration of in vitro replication by a mammalian replication system (Alberts et al. 2014; Nasmyth & Schleiffer 2004).

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Eventually, using Saccharomyces cerevisiae as the model organism for investigating DNA replication in eukaryotes, all the components, enzymes and sequence of events involved in DNA replication were analyzed. It was then observed that the fundamental features of DNA replication mechanism were similar in eukaryotes and prokaryotes (Smith & Szathmary 1997; Alberts et al. 2014; Lodish et al. 2012). It is now known that organization of the replication fork and participation of macromolecular complexes has remained conserved across all domains of life. The question arises then, that why do we need to study eukaryotic DNA replication as a separate topic of study? Well, the answer is, in spite of the conserved features, the eukaryotic DNA replication machinery is far more complex and there exist certain striking differences such as, the consensus sequences are different, the rate of replication is slower, the number of components and enzymes involved is much greater in eukaryotes and these components are analogous to their bacterial counterparts but not identical (Alberts et al. 2014; Nasmyth & Schleiffer 2004).

A. Origin of Replication and Pre-replication Complex

Origin of replication: The specific region(s) on the chromosome where unwinding of DNA duplex is initiated thus triggering DNA replication, is termed as the Origin of Replication. Similar to prokaryotes, the replication origin in eukaryotes is also characterized by a conserved consensus sequence. However, eukaryotic systems show certain significant deviations from prokaryotic replication origin.

In eukaryotes, replication of DNA occurs only during the S-phase (or synthetic phase) of the cell cycle and the euchromatin is replicated during early S-phase while heterochromatin replicates during late S-phase (Alberts et al. 2014; Lodish et al. 2012). In contrast to a single on the bacterial chromosome, the eukaryotic genome is characterized by multiple functional replicons. At any given time during the S-phase, multiple replication origins are simultaneously operative throughout the eukaryotic genome and also on every single chromosome. In the early 1970’s, Huberman and Riggs for the first time, demonstrated the occurrence of multiple bidirectional replication origins in the mammalian chromosomes which remain scattered at approximately 100 kb intervals (Hamlin 1992). It is noteworthy that while the eukaryotic genome is many folds larger in size than prokaryotic genome, the rate of progression of the replication fork in eukaryotes has been demonstrated to be only about 50 per second (in contrast to ~1000 nucleotides per second in prokaryotes) (Benard et al. 2007). It is speculated that the extremely slow movement of replication fork in eukaryotes may be attributed to the time lag required to unfold the multiple layers of condensed packaging of DNA in eukaryotic chromosome. The possible reason of formation of multiple replicons in eukaryotes could be to counter the slow progression of replication fork and achieve complete genome replication within the stipulated time during the cell cycle (Alberts et al. 2014; Lodish et al. 2012).

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ARS: It is now known with certainty that the replication origin in prokaryotes and lower eukaryotes spreads across several hundred nucleotides and is Fig. 2.1: Example of ARS – a core consensus sequence characterized by stretches of multiple at replication origin in eukaryotic chromosome consensus sequences. In higher eukaryotes however, the replication origin is marked by much shorter AT-rich sequence of nucleotides known as the Autonomously Replicating Sequence (ARS) (Fig. 2.1) (Vindigni et al. n.d.). A core consensus sequence of 11 base pairs may act as the ARS in many higher eukaryotes including mammals. Other known AT-rich consensus sequences such as the Origin Enriched Sequences (ors8, ors12) have been identified in the DNA replication bubble at the beginning of the S- phase (Zannis-Hadjopoulos et al. 1992; Mah et al. 1992).

Since 1960’s till date, with advancement in experimental techniques and technology, origin of replication has been investigated in scores of eukaryotic genomes. All these studies point to certain common characteristic features of the DNA sequences functioning as the replication origin (Alberts et al. 2014; Baranovskiy & Tahirov 2017; Lodish et al. 2012):

1) Origins of replication are found to be invariably rich in A and T bases, making the localized denaturation DNA duplex easier compared to the G-C rich sequences due to an additional hydrogen bonding in the latter case 2) The DNA sequence in the replication origin contains a binding site for the macromolecular, multi-subunit initiator called the Origin Recognition Complex (ORC); 3) DNA sequence in the origin of replication contains at least one binding site for the ORC loader protein which facilitates binding of the ORC onto the DNA.

Pre-replication complex: A multi-protein, multi-subunit, macromolecular complex which assembles at the origin of replication to initiate DNA replication is known as the pre-replication complex. Although pre-RCs are known to occur as a conserved feature across all living organisms, the pre-RC of eukaryotes is by far the most complex and larger than that of the lower organisms. The assembly of the pre-RCs at the origin is a precisely coordinated event. It is speculated that the time and sequence of assembly of different pre-RCs at their respective origins of replication and the sequential loading of are critically regulated and used by the cell as an internal control mechanism to ensure that the entire genome is replicated only once during the cell cycle and that the adjacent replication bubbles do not overlap (Okuno 2001; Takisawa et al. 2000).

Components of Pre-RC in Eukaryotes: The most complex and elaborate pre-RCs are known to occur in the eukaryotes. Each of the multiple subunits of pre-RC discharges a defined and significant functional role in initiation of DNA replication.

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Evidences suggest that the Origin Recognition Complex, a hetero- hexamer of six different subunits (subunits 1 to 6) is the first component of pre-RC that directly recognizes and binds onto the replication origin (Jelena & Drag 2011). ORC has been shown to have a DNA dependent ATPase activity that channelizes energy by ATP hydrolysis for the subsequent events of pre- RC assembly and initiation of DNA replication. Sequential recruitment of other Fig. 2.2: Assembly of pre-Replication Complex in Eukaryotes components of pre-RC Source: https://www.nature.com/nrm/journal/v11/n10/box/nrm2976_BX1.html (Méchali 2010) (Table 2.1) with the final loading of hetero-hexameric Mini Chromosome Maintenance proteins (MCM2 to 7) (Jelena & Drag 2011; Fragkos et al. 2015) that functions as the ATPase dependent DNA helicase marks the committed initiation of DNA replication and completes the pre replication complex assembly (Takisawa et al. 2000; Méchali 2010).

Name of the component Function ORC1–6  Initiates pre-RC formation;  ATPase activity Facilitates loading of MCM complex (Cell Division Control protein 6 homologue) CDT1  Facilitates loading of MCM complex (CDC10 Dependent Transcript 1)  Interacts with CDC6 MCM2–7 ATPase dependent DNA helicase DDK Phosphorylates and activates MCM complex (DBF4-Dependent ) CDKs Activation of Helicase CDC45  Helicase activation (Cell Division Control protein 45 homologue)  Initiates replisome assembly Geminin  Inhibitor of pre-RC  Inhibits formation of new pre-RCs by binding to CDT1

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B. Primosome and Replisome Complexes

Primosome Complex: In the simplest terms, primosome is the multi-protein complex responsible for “priming” or creating the RNA primers during DNA replication. The functional role of primosome is similar in eukaryotes and prokaryotes; the deviation is in the individual components and subunits. None of the known DNA polymerases possess a de novo polymerizing ability and hence require a pre-existing polynucleotide chain with a free 3’ OH position. This requirement is fulfilled by the primosome which includes a DNA dependent RNA polymerase activity in one of its subunits. Precisely timed assembly and activity of primosome is critical for successful, accurate and complete DNA replication in eukaryotes (Alberts et al. 2014; Baranovskiy & Tahirov 2017).

The eukaryotic primosome is a complex of four identifiable subunits derived from two distinct enzymes: DNA (DNA-dependent RNA polymerase) and DNA polymerase (Pol). Initiation of replication on both strands of the DNA duplex, the leading and the lagging strand, begins with the synthesis of a short stretch of DNA templated RNA primer by DNA primase that forms a hybrid RNA/DNA hetero- duplex. Thus functional involvement of primosome is required for “priming” or synthesis of the RNA primer on both the strands which then stimulates assembly of the components of replisome complex including the replicative DNA polymerases. At every replication bubble, primosome, or the DNA primase, synthesizes RNA primer only once on the leading strand, but multiple times on the lagging strand for synthesis of individual discontinuous which are ~165 nucleotides long in eukaryotes (Okazaki 2017). During post synthesis editing and maturation of the lagging DNA strand, the RNA primers and short portions of the newly synthesized DNA are removed by an exonuclease; and the remaining Okazaki fragments are extended by DNA pol which fills the gap by adding complementary deoxyribonucleotide dictated by base pairing with the template strand.

Human primosome: Let us understand the structure and function of human

Fig. 2.3: Schematic representation of the domain organization in the human primosome Source: Review | Elaborated Action of the Human Primosome; Baranovskiy and Tahirov; Genes | 2017

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primosome and the stepwise sequence of events leading to RNA primer synthesis during DNA replication in humans. The salient features of human primosome (Baranovskiy & Tahirov 2017):

 Size: 340 kDa four subunit protein complex  Components and domain organization (Fig. 2.3): DNA primase: DNA dependent RNA polymerase o 50 kDa catalytic subunit o 59 kDa regulatory subunit o Generates only 9 long RNA primers by three defined steps of initiation, elongation and termination DNA polymerase-: B-family, 5’ 3’ DNA polymerase o 166 kDa catalytic subunit o 66 kDa accessory subunit o Polyemerizes RNA/DNA duplex and DNA/DNA duplex with similar efficiency o Begins extension of 9-mer RNA primer by intramolecular functional switching with DNA primase component of primosome o Extends DNA templated RNA primer to 30-35 bases long RNA/DNA chimeric primer by adding dNTPs complementary to the template strand

Human primosome undergoes a series of intramolecular conformational changes and structural modulations (Fig. 2.4) to achieve seamless synthesis of RNA/DNA hybrid duplex which is then made available to DNA pol  and DNA pol . The entire cycle of events occurs only once on the leading strand and at repeated intervals on

Fig. 2.4: Series of conformational changes in human primosome during chimeric primer synthesis Source: Review | Elaborated Action of the Human Primosome; Baranovskiy and Tahirov; Genes | 2017

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the lagging strand for priming each Okazaki fragment. The dynamic arrangement of various domains in the human primosome complex gives rise to three distinct functional centres: RNA-polymerizing center, DNA-polymerizing center and regulatory center for intramolecular translocation from primase to DNA pol (Baranovskiy & Tahirov 2017).

Replisome Complex: Genome replication in eukaryotes is a closely coordinated series of multistep events. Complete and accurate DNA replication in eukaryotes involves a sequential and regulated assembly of several multiprotein complexes. Replisome is the large multi- protein, multi-subunit macromolecular machinery that assembles after the primosome (Prakash & Borgstahl 2012) in order to carry out the subsequent Fig. 2.5: Schematic representation of eukaryotic replisome complex steps of DNA replication. The Source: SnapShot: The Replisome; Nina Y. Yao and Mike O’Donnell | Cell, 2010 combined action of pre-RC and primosome at the origin of replication on the eukaryotic DNA induces local unwinding of the DNA duplex forming a nascent replication bubble which is flanked on either side by a replication fork. Each replication fork is simultaneously stabilized by assembly of a replisome complex (Fig. 2.5) which maintains the unwound state of DNA double helix and also facilitates movement of the replication fork along the DNA duplex away from the origin of replication. Thus, the DNA duplex continues to unwind into two single stranded individual templates both of which are subsequently copied in the 5′ to 3′ direction to form two new DNA duplex fragments per replication bubble. Comparative analyses have revealed that the fundamental components of the replisome are evolutionarily conserved across , , , and eukaryotes (Yao & O’Donnell 2010)(Sun et al. 2015). The eukaryotic replisome assembly begins with the loading of the CMG (Cdc45, Mcm2–7, GINS) helicase (ATP dependent DNA helicase) at the replication fork (Georgescu et al. 2017). Chief components of eukaryotic replisome complex are tabulated in Table 2.2:

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Name of the component Functional role

MCM2-7/CMG  Component of the CMG helicase complex  Hetero-hexameric ring like protein complex  Translocates in the 3′ to 5′ direction along the leading strand to separate the duplex DNA GINS, Cdc45/CMG  Other components of the CMG helicase complex  Act as helicase assistant DNA pol ε  Synthesis of leading strand  5’ 3’ polymerase  3’5’ proofreading exonuclease DNA pol δ  Synthesis of lagging strand  5’ 3’ polymerase  3’5’ proofreading exonuclease DNA pol β  5’ 3’ polymerase  Base excision repair Pol -Primase Primosome complex for RNA priming on the lagging strand PCNA Homotrimeric protein complex acts as the sliding (Proliferating Cell Nuclear Antigen) DNA clamp RFC Clamp loader protein () RPA1  Single strand binding protein ()  Prevents rewinding of DNA duplex

C. Eukaryotic DNA Polymerase

The eukaryotic DNA polymerases have been less investigated in general, than their prokaryotic counterparts. The most well studied DNA replication machinery and DNA polymerase among eukaryotes are those of the Saccharomyces cerevisiae and a few mammals including humans and mice. All the available evidences suggest that the overall functioning and mechanism of polymerization is conserved between the eukaryotic and prokaryotic DNA polymerases (Wang 2002; H. bscher 2010; Walsh & Eckert 2013; Cotterill & Kearsey 2014). Both these groups of DNA polymerases function as DNA dependent DNA polymerase; synthesize DNA only in 5’3’ direction; cannot begin de novo DNA synthesis and invariably require an existing polynucleotide chain with a free 3’ OH position to begin polymerizing activity.

So far, at least fifteen different DNA polymerases have been discovered in different eukaryotic systems which may be grouped into different families of polymerases. The main replicative polymerases involved in eukaryotic DNA replication are DNA pol α, DNA pol ε and DNA pol δ, all three belonging to the polymerase family-B, and

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are encoded by POLA1 & POLA2 genes, POLE and POLD1 gene respectively (Sarasin et al. 1983; Burgers 1998).

Six other types of DNA polymerases known in eukaryotes make up the polymerase family X which mostly occurs in vertebrates and rarely in plants and fungi. Family X includes DNA pol β (encoded by POLB gene), Pol λ (encoded by POLL gene), Pol σ, Pol μ (encoded by POLM gene) and Pol ζ forms of polymerase and Terminal deoxynucleotidyl (TdT) which is known to occur only in the lymphoid tissue (Cotterill & Kearsey 2014; Walsh & Eckert 2013).

Family A polymerases of eukaryotes includes the Telomerase and the Pol γ and Pol θ (encoded by POLQ gene) forms, family Y polymerases include Pol η, Pol ι and Pol κ forms. A special type of is also reported in eukaryotes (Hübscher 2005; Walsh & Eckert 2013).

A functional summary of important eukaryotic DNA polymerases is as follows:

 DNA pol ε: POLE1, POLE2, POLE3, POLE4 subunits o High o 5’  3’ polymerase o 3’  5’ proofreading exonuclease o Synthesis of leading strand  DNA pol δ: POLD1, POLD2, POLD3, POLD4 subunits o High processivity o 5’  3’ polymerase o 3’  5’ proofreading exonuclease o Synthesis of lagging strand  DNA pol α: POLA1 & POLA2 subunits o 5’  3’ polymerase o Initiates replication & extends RNA primers o No exonuclease activity  DNA pol β: PRIM1 & PRIM2 subunits o 5’  3’ polymerase o Low fidelity replication o Base excision repair and DNA damage repair  DNA pol γ: o 5’  3’ polymerase o 3’ 5’ exonuclease o Mitochondrial genome replication o Chloroplast genome replication  DNA pol λ: repairing DNA double strand breaks caused by hydrogen peroxide  DNA pol μ: repairing DNA double strand breaks caused by ionizing radiations  DNA pol θ: o DNA-dependent ATPase in human cell

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o DNA inter-strand crosslink repair o Double-strand breaks by alternative end-joining repair o Promotes DNA replication by resolving oxidative lesions and unrepaired lesions  DNA pol κ : no exonuclease activity, required for attachment of Cohesin  Telomerase: Replication of , the terminal ends of linear chromosomes  TdT: Adds non-templated nucleotides to provide diversity  Reverse transcriptase: RNA dependent DNA polymerase

D. Telomere and Telomerase

Telomere: DNA replication on the lagging strand occurs in the form of discontinuous Okazaki fragments each beginning with a RNA primer. During maturation of the strand, the RNA primers are edited out and replaced with DNA bases before the ligase finally seals the gap between adjacent Okazaki fragments to form the intact lagging strand. But when the replication fork reaches the terminal part of the lagging strand on the linear chromosome of eukaryotes, there is no scope to remove the RNA primer due to obvious physical constraints. Thus the eukaryotic cells have evolved a special mechanism to prevent the loss of DNA from the terminal portions of the linear chromosomes at the end of each round of DNA replication (Sampathi & Chai 2011; Brevet et al. 1999; Alberts et al. 2014).

The ends of the linear chromosomes are made of special nucleotide sequences called Telomeres that have been shown to be conserved across all groups of eukaryotes from protozoans, fungi, plants and mammals. Telomeres are known to contain multiple repeats of short conserved G-rich clusters which do not code for any particular gene, thus protecting any gene from getting deleted as cells continue to divide. Human telomeres are characterized by more than thousand repeats of a six sequence [-GGGATT-]. It is now known that telomeres prevent fusion of wrong ends of broken chromosomes, thus providing stability to chromosomes (Kierszenbaum 2000; Sfeir et al. 2005; Eisenstein 2011).

Telomerase and Telomere replication: There exists a specialized mechanism for replication of these specialized sequences which was discovered and elucidated by the Nobel Laureate Elizabeth Blackburn. Elizabeth Blackburn discovered the Fig. 2.6: Elizabeth Blackburn received the Nobel characteristic DNA in the telomeres (1980) Prize for Medicine and Physiology in 2009 for and later, together with Jack Szostak her discovery of telomerase and its action Source: http://philschatz.com/biology- book/contents/m44517.html

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established the importance and mode of replication of telomeres (1982). Further Elizabeth and Carol Greider together discovered the enzyme telomerase that is mainly responsible for telomere replication (1984) and also established its mode of action (Blackburn 1986; Prescott & Blackburn 2002; Blackburn 2010).

Telomere replication is a sophisticated and closely regulated series of events that includes combined and concerted action of a complex of proteins and enzymes. Fig 2.7: Activity of Telomerase in eukaryotic DNA replication http://philschatz.com/biology-book/contents/m44517.html Telomere replication machinery comprises of sequence specific DNA binding proteins which upon binding to the telomere, stimulate the assembly of the enzyme complex called telomerase (David 2012; Gilson & Géli 2007). Telomerase, the enzyme that helps in replicating the telomere of lagging strand contains a built-in RNA template complementary to the 3’ overhang of the lagging strand telomere and a catalytic part which has Reverse transcriptase like action so that it performs RNA dependent DNA polymerization to generate the lagging strand telomere sequence at the end of DNA replication on both terminals of the linear chromosomes (Fig. 2.7). Human telomerase complex is known to comprise of two molecules each of human telomerase reverse transcriptase (TERT), telomerase RNA (TR or TERC), and (DKC1) (Mitchell & Collins 2000; Bachand & Autexier 2001; Kumaki et al. 2001).

III. DNA REPLICATION MECHANISM IN EUKARYOTES

The entire life cycle of any organism and all vital activities essential for survival, sustenance, growth and proliferation of the organism are known to be genetically programmed. This genetic information contained in the genome of the organism must be completely replicated and faithfully transmitted to subsequent generation for maintenance of the species. Thus for all organisms, grouped into any domain of life, DNA replication is a pivotal event in deciding the fate of the cell and hence fate of the organism, both in unicellular and multicellular organisms. Eukaryotes also are no exception to this fact.

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Further, we have compelling evidence indicating that the overall mechanism of DNA replication has remained conserved through the course of evolution across all domains of life, prokaryotes and eukaryotes. However, in eukaryotes the DNA replication happens to be a more complex and elaborate sequence of events which spans over much longer duration of time.

A. General Model of DNA Replication in Eukaryotes

DNA replication in eukaryotes, by and large, is semi-conservative, semi- discontinuous and bidirectional in nature (Iwamura et al. 1982; Filner 1965; Dhingra & Kaplan 2016). The complex multistep sequence of events is accomplished in three distinct phases: initiation, elongation and termination. Replication of DNA is achieved by complementary base pairing where an Adenine pairs with Thymine by forming two hydrogen bonds and Guanine pairs with Cytosine by forming three hydrogen bonds (Gresh & Šponer 1999; Dhingra & Kaplan 2016). This is the regular Watson-Crick base pairing which is largely responsible for holding the two strands in DNA duplex together over extensive lengths. At the time of replication, the DNA duplex unwinds and opens up by localized denaturation of hydrogen bonds catalyzed by the ATP dependent DNA helicase (CMG helicase complex). Thus two individual parent strands are exposed as the template for DNA-templated DNA polymerization, where, again, the sequence of nucleotides being polymerized into the new DNA strand is governed by Watson-Crick base pairing which in turn is directed by the nucleotide sequence of the parent template strand.

The main replicative polymerase in eukaryotes are DNA polymerase // (Pol //) (Walsh & Eckert 2013). Similar to the replicative DNA polymerases of E. coli, eukaryotic DNA polymerases also lack de novo synthesis ability; exhibit only 5’  3’ polymerase activity and can add a new base only to the free 3'-OH group available at the free 3’ end of the growing polynucleotide chain (Anon 2010; Matsukage et al. 1983). The biochemical mechanism of polymerization is similar in eukaryotes and prokaryotes. Polymerization of free nucleotide by Pol // involves a nucleophilic attack by the 3’-OH group of the nucleotide at the at the 3’ end of the growing strand on the -P of the incoming dNTP, complementary to the next base in the template strand. This is accompanied by elimination of the pyrophosphate. Almost every step in the multistep event of DNA polymerization requires energy which is derived from hydrolysis of dNTPs and the resulting pyrophosphates (Berg et al. 2015; Chatterjea 2012; Alberts et al. 2014). At the onset, the DNA helix is unwound and prepared for synthesis by the combined action of pre-replication complex, DNA helicase and the primosome. Since DNA polymerase lacks intrinsic de novo synthesis ability, it invariably requires an existing polynucleotide chain with free 3’-OH terminal upon which the DNA polymerase can act as extension polymerase. But the cells contain certain RNA polymerases which are equipped with the de novo synthesis ability (Wang 2002; Alberts et al. 2014; Krebs et al. 2017). A specialized DNA dependent RNA polymerase, known as the

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Primase executes the first step in the initiation of synthesis of new DNA strand, known as “Priming” during which the Primase synthesizes a short nine nucleotide stretch of RNA primer complementary to the exposed 3’-OH terminal of the template strand. This provides the free 3'-OH group required to initiate replication. Thereafter the extension polymerase Pol  takes over the polymerizing activity by intramolecular rearrangement within the primosome that shifts the template from the primase to the Pol  which extends the RNA primer by adding complementary DNA bases. Thus RNA primer and a chimeric RNA/DNA primer is formed which is then extended by normal polymerization action of replicative polymerases Pol /. (Baranovskiy & Tahirov 2017).

On the leading strand, only a single RNA primer is needed, and DNA is synthesized continuously in 5’  3’ direction by Pol  . On the lagging strand, DNA is synthesized in a discontinuous manner by formation of multiple short stretches of polynucleotides called Okazaki fragments, each synthesized in 5’3’ direction. Each Okazaki fragment synthesis requires its own primer and the combined repetitive action of Primase, Pol  and Pol . The final product however, does not have RNA stretches in it. At the time of maturation the RNA primers and a small part of the initially added DNA bases are excised out by the 5' to 3' exonuclease action of FEN1 and RNAse H (Rydberg & Game 2002). The gaps in the lagging DNA strand resulting from the removal of the RNA primer are filled in by the 5’  3’ polymerase action of DNA Pol  and the nicks are joined by DNA ligase thus forming an intact lagging strand.

DNA replication in eukaryotes initiates at multiple origins and proceeds through multiple replicons that are operative adjacent to each other along the length of the chromosome. The exact mechanism of termination of individual replicons in eukaryotes is not yet clearly deciphered and no specific termination sequence for individual replicons has yet been identified in eukaryotic genomes. It is thus speculated that linear DNA replication continues until the two replication forks belonging to two adjacent expanding replication bubbles approach and meet each other after which termination occurs in a more or less unregulated manner or in a manner whose mechanism of regulation is yet to be identified. Another theory suggests that as the two adjacent replicons approach each other, DNA polymerase halts its polymerizing action when it reaches a segment of DNA that has already been replicated. In either case DNA polymerase is unable to catalyze phosphodiester bond formation between the adjacent newly replicated DNA fragments and it drops off (Dewar et al. 2015; Huberman 1974; Z. et al. 2011) The nicks are sealed by ATP dependent DNA ligase to form intact leading and lagging strands on both the new DNA duplexes and thus the termination of individual replicons is achieved (Zimmerman & Levin 1975).

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B. Enzymes Involved In Eukaryotic DNA Replication

As it has been repeatedly emphasized in this module, DNA replication in eukaryotes is an extremely complex, well-coordinated and closely regulated multi step sequence of events. The living cells are known to use proteins and enzymes as the molecular switches for regulating all cellular activities. Each step of DNA replication is driven and regulated by complex macromolecular machinery largely comprised of enzymes and proteins. In the preceding sections, most of the eukaryotic enzymes and other proteins forming different macromolecular complexes and involved in individual DNA replication steps in eukaryotic system have already been discussed.

Here we present a snapshot of events (Fig. 3.1) and summary of the important enzymes involved in eukaryotic DNA replication.

Summary of Enzymes:

1) Eukaryotic DNA polymerase: a. DNA pol α (POLA1, POLA2) b. DNA pol β (PRIM1, PRIM2) c. DNA pol γ d. DNA pol δ (POLD1, POLD2, POLD3, POLD4): synthesis of leading strand e. DNA pol ε (POLE1, POLE2, POLE3, POLE4): synthesis of lagging strand 2) DNA helicase: ATP dependent unwinding of the DNA helix

3) DNA primase: DNA-dependent RNA polymerase- synthesizes chimeric RNA- DNA primers

4) : Releases topological stress created in the DNA duplex by the bidirectional expansion of replication bubble.

5) : A cytoplasmic protein, absolutely essential for triggering DNA replication at the origin, but can access the chromosome only during a certain stage of the S-phase, ensures that each origin is triggered only once during the cell cycle.

6) PCNA (Proliferating Cell Nuclear Antigen): sliding clamp protein, holds the DNA pol in place

7) Replication factor C (RFC1)

8) (FEN1)

9) Replication protein A (RPA1)

10) RNase H: removes the RNA primer, which is then replaced by deoxy nucleotides

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11) DNA ligase: forms the phosphodiester bonds to seal the gaps between Okazaki fragments

12) Telomerase

Fig. 3.1: Snapshot of events and components of DNA replication machinery in eukaryotes Source: http://reasonandscience.heavenforum.org/t1849-dna-replication-of-prokaryotes| Credit: Kenhisa Lab

C. Steps Involved in DNA Replication in Eukaryotes

Initiation: DNA replication in eukaryotes occurs only during the S-phase of cell cycle (Fig. 3.2) when a speculated cytoplasmic protein termed as ‘Licensing factor’ translocates into the nucleus and reaches the Origin of Replication to trigger DNA replication. This is followed by assembly of pre-replication complex, completed with loading of two copies of DNA helicase at Origin. ATP dependent unwinding of DNA double helix by DNA Helicase leads to formation of a replication bubble and marks the commencement of a long series of cascading events leading to DNA replication (Fig 3.3). Clusters of nearly 20-50 replicons initiate simultaneously at their respective starts sites or ‘origins’ distributed throughout the length of linear chromosome.

Fig. 3.2: Assembly of pre-RC leading to Initiation Complex, Primosome and Replisome. Source: https://en.wikipedia.org/wiki/Eukaryotic_DNA_replication#/me dia/File:Pre-replicative_complex.JPG

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Initiation phase ends with assembly of Primosome complex at the origin followed by synthesis of chimeric RNA-DNA primer by Primase and elongation of the hybrid primer by Pol.

Fig 3.3: Formation of replication bubble and replication fork at origin Source: https://www.boundless.com/biology/textbooks/boundless-biology-textbook/- structure-and-function-14/dna-replication-101/dna-replication-in-eukaryotes-437-12941/

Elongation: The replication bubble is flanked by a replication fork on either side which is stabilized by assembly of a Replisome Complex (Fig 3.4). This macromolecular machinery is responsible for elongation of the new DNA strands which occurs invariably in the 5’  3’ direction in a DNA templated DNA polymerization pattern. In the eukaryotic cell, the replication fork progresses at a modest rate of 50 nucleotides per second. This slow rate of replication in eukaryotic DNA may be attributed to the time lag required to unpack the multiple layers of compaction of the eukaryotic genome (Nasheuer et al. n.d.; Leman & Noguchi 2013). The sequence of the incoming nucleotide in the elongating polynucleotide chain is directed by complementary base pairing with the nucleotide sequence in the template strand. Leading strand is replicated seamlessly in 5’  3’ direction while the lagging strand is synthesized in the form of discontinuous

Fig. 3.4: Activities and events during elongation of DNA replication | Credits: Bumphrey Source: https://en.wikipedia.org/wiki/Eukaryotic_DNA_replication#/media/File:Replication_complex.png

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Okazaki fragments which are later backstitched to produce the intact lagging strand. The overall mechanism (Fig. 3.5) has been discussed in a previous section of this module

Fig. 3.5: Eukaryotic replisome complex and events during elongation of DNA replication https://en.wikipedia.org/wiki/Eukaryotic_DNA_replication

Termination: During DNA replication in eukaryotic cells, multiple adjacent replicons are operative simultaneously along the length of linear chromosomes. When two adjacent replicons progressing towards each other collide, the DNA polymerase drops off as it is unable to replicate those segments of DNA which have already been replicated. The exact mechanism and regulation of this step still remains unclear and requires more investigation. The final sealing of the newly synthesized adjacent DNA segments on leading and lagging strands is mediated by ATP dependent DNA Ligase that joins the nicks to form two individual mature DNA duplexes.

The terminal portions of the linear chromosomes contain specialized sequences called telomeres and the replication of telomeres is mediated by the Telomerase Complex (Fig. 3.6) (Gilson & Géli 2007). This happens to be a RNA-protein complex with an inbuilt RNA template complementary to the telomere sequence of the lagging strand and synthesizes the telomeres in a Reverse Transcriptase (RNA dependent DNA polymerase) like action (Mitchell 2001).

Telomere replication reportedly has a role in determining the life span of an organism and gradually reducing telomerase activity is known to correlate with and ageing. Anomalous replication of telomere and telomerase dysregulation in humans is known to be associated with tumorigenesis and related pathological conditions (Fig. 3.7) (Schluth-Bolard et al. 2011)

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Fig. 3.6: Replication of telomere terminals by telomerase Source: https://en.wikipedia.org/wiki/Telomerase#/media/File:Working_principle_of_telomerase.png

Fig. 3.7: Correlation of telomere replication and human diseases Source: http://atlasgeneticsoncology.org/Deep/SubTelomereID20025.html

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IV. FIDELITY OF DNA REPLICATION IN EUKARYOTES

The eukaryotic genome is an enormous reservoir of all the essential information required for survival and sustenance of any organism. The importance of accurate, precise and singular replication of the entire eukaryotic genome before every cell division and its faithful distribution into two daughter cells can never be over emphasized. Inspite of the enormous size of the eukaryotic genome and its complicated packaging, every eukaryotic cell has inherent mechanisms to ensure that the DNA is replicated with extreme precision and high fidelity and it is done only once per cell cycle.

Fig. 4.1: Graphic representation of the various means by which DNA replication can be modulated in a cell Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3639319/pdf/nihms43702.pdf

In all eukaryotic cells including humans, proofreading activity of the replication machinery is the primary keeper of fidelity of replicative DNA polymerases. It is well documented that in the course of DNA replication, specific proofreading polymerases work in concert with replicative, non-proofreading polymerases to ensure faithful and precise DNA replication of the entire genome across all the linear chromosomes (McCulloch & Kunkel 2008; Alberts et al. 2014). Experimental assays indicate that the average error rate by replicative DNA polymerases in eukaryotes is no more than once every 104 to 105 bases incorporated. This error if allowed to remain uncorrected to eventually result in accumulation of approximately 104 errors per genome replication in a typical mammalian cell, which is a potentially lethal threat. The errors caused during replication of mammalian genome by DNA polymerase is rectified by combined activity of the 3’  5’ proofreading exonuclease and that of the post-replicative strand directed mismatch repair mechanism (Fig. 4.1)

Determinants of Replication Fidelity: Combined actions of the A, B and partially X family DNA polymerases are mainly responsible for the three known inherent

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Fig. 4.2: Graphic representation of relative contribution levels of the three main components of replication fidelity Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3639319/pdf/nihms43702.pdf

mechanisms that ensure high fidelity of the DNA replication machinery in eukaryotic cells (Fig. 4.2). These three steps collectively (Table 4.1) give rise to in vivo or error rate lower than ~1 ×10−9, i.e., less than one error for every billion (or more) base pairs copied

. Replication step Errors per nucleotide 5’3’ Polymerization 1 in 105 3’ 5’ Exonucleolytic proofreading 1 in 102 Strand directed mismatch repair 1 in 102 Combined 1 in 109

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V. SUMMARY

Particulars Eukaryotes Prokaryotes Time of replication S phase End of Cell Cycle

Site of replication Nucleus Cytoplasm

Template used Pre-existing DNA Pre-existing DNA DNA polymerase DNA polymerase: I, II Enzymes Involved //// and III. Direction of 5’-3’ direction 5’-3’ direction Replication Mechanism More Complex Simple Active Replication Multiple, ARS One, oriC sites Replicons Multiple Single Replication rate per Slow, 50-100 bases Rapid, 500-1000 bases second Okazaki Fragments 100-200 residue long 1000-2000 residue long

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DNA replication in eukaryotes is semi-conservative, semi-discontinuous and bidirectional in nature that occurs only during S-phase of cell cycle

Basic mechanism of DNA replication is conserved in prokaryotes and eukaryotes , but it is more complex and elaborate in eukaryotes.

DNA replication in eukaryotes is a closely coordinated phenomenon that initiates simultaneously at multiple origins of replication.

Many of the cell cycle related disorders in humans are associated with disruption of the checkpoints of DNA Replication

p53 and p21 mediated checkpoints of S-phase in eukaryotic cell cycle are known to be disrupted in many cases of carcinogenesis

Knowledge of such and other conserved essential genes would serve as novel targets for genome engineering and molecular therapy

Telomerase dysfunction that correlates with many tumorigenic pathologies, is being targeted as a molecule of choice for anti cancer therapy

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