DNA is the universal genetic material. DNA replication in prokaryotes is

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Biotechnology Genetic Engineering and Recombinant DNA Technology

DNA Replication in Prokaryotes Page 1 of 28

Paper No. : 04 Genetic Engineering and Recombinant DNA Technology Module : 08 DNA Replication in Prokaryotes

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 Content Reviwer: Dr Mohan Chandra Joshi, Assistant Professor,

Jamia Millia Islamia, New Delhi

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DNA Replication in Prokaryotes Page 2 of 28 Description of Module

Subject Name Biotechnology

Paper Name Genetic Engineering and Recombinant DNA Technology Module

Name/Title

DNA Replication in Prokaryotes

Module Id 08

Pre-requisites Basics of DNA structure and composition Objectives ● Fundamentals of DNA Replication

● DNA Replication in Prokaryotes: Enzymes and components

● Steps In DNA Replication

● Fidelity of DNA Replication

Keywords Semi-conservative, DNA polymerase, Primosome, Replisome, Proof-reading

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INDEX

LEARNING OBJECTIVES ABOUT THE MODULE I. INTRODUCTION

A. DNA – From “Nuclein” to the Deoxy-ribonucleic Acid B. DNA as the Universal Genetic Material

C. DNA Structure: Journey to the Phenomenal Double-helix II. FUNDAMENTALS OF DNA REPLICATION

A. Suggested Models of DNA Replication

B. Semi-Conservative Nature of DNA replication C. Characteristic Features of DNA Replication III. DNA REPLICATION IN PROKARYOTES

A. Enzymes Involved In Bacterial DNA Replication

B. Origin of Replication, Primosome Complex and Replisome Complex in Bacteria

C. Steps Involved in DNA Replication in Prokaryotes IV. ^FIDELITYOF DNA REPLICATION IN PROKARYOTES V. SUMMARY

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DNA Replication in Prokaryotes Page 5 of 28 DNA was established as the universal genetic material towards the

middle of the 20^th century. The illustration of double helical structure of DNA, semiconservative nature of DNA replication and later the discovery of DNA pol I, unlocked a whole new dimension of research and studies in modern biology and laid the foundations for the advanced disciplines like Genetic Engineering and Recombinant DNA Technology. But all of these could never have been possible without a detailed insight into the molecular organization and mechanism of replication DNA. The main focus of the present module is to present a detailed account of “DNA Replication in Prokaryotes” that are argued to be the ‘original replicators’. The introductory part of the module describes all you need to know about DNA and guides you through a timeline study to help you understand how DNA was established as the universal genetic material. Thereafter we attempt to outline the overall nature and fundamentals of DNA replication in general. DNA replication in prokaryotes, in particular, is dealt with in the subsequent segment where we focus on the enzymes and components of prokaryotic DNA replication machinery. The subsequent section of the module takes you through a detailed discussion about the steps in prokaryotic DNA replication and the module concludes with an understanding of the fidelity of prokaryotic DNA Replication.

LEARNING OBJECTIVES:

● DNA as the Universal Genetic Material

● Fundamentals of DNA Replication

● DNA Replication in Prokaryotes: Enzymes and components

● Steps In DNA Replication

● Fidelity of DNA Replication

● Summary

ABOUT THE MODULE

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DNA Replication in Prokaryotes Page 6 of 28 I. INTRODUCTION

he evolution and establishment of ‘Molecular Biology’ as an independent discipline in Biotechnology stems from the concept of the ‘Central Dogma of Life’. Eventually, Molecular Biology laid the foundations of Recombinant DNA Technology and Genetic Engineering. One can never ignore the contribution of a strong fundamental for raising a superstructure. Thus the knowledge of Molecular Biology is of paramount importance in comprehending the fundamentals Recombinant DNA Technology.

In the most simplified terms, the ‘central dogma’

states that DNA dictates synthesis of RNA (Transcription) and the RNA dictates synthesis of proteins (Translation). This representation, albeit, is oversimplified and severely understates the substantial functional roles of proteins in gene expression and gene regulation (Lodish et al.

2012; Alberts et al. 2014). The proteins execute exceptional functional diversity as the molecular triggers and regulators, cellular catalysts and components of structural framework and metabolic pathways.

The information for coding of proteins ultimately is packed in the form of a database of nucleotide

sequences known as the ‘genetic code’, which in turn is embedded in the genetic material of all organisms. Thus, faithful replication of genetic material followed by its accurate distribution and propagation from one generation to the following is of pivotal importance for growth and survival of organisms. Prokaryotes, arguably the earliest known living organisms and the original replicators, provide the ideal platform and model to have a detailed insight into the mechanism and mode of replication of genetic material (De la Bédoyère 2005; Allison 2011), which today is known to be the Deoxyribonucleic Acid or the DNA. In the present module, our focus of discussion will be “DNA Replication in Prokaryotes” where we shall discuss all the components, mechanism and regulation of DNA replication considering E. coli, one of the most well studied systems, as the model organism. But was DNA always known to be the (only) genetic material? If not, how did DNA come to be accepted as the ‘universal genetic material’? Have you ever wondered, what made evolution and natural selection select DNA as the genetic material for all living organisms while there exist several other biomolecules? Before studying the mechanism of replication of DNA, we must first answer a few of these questions. Let us begin with a comprehensive look into the timeline of events that established DNA as the genetic material and illustrated the structure and mode of replication of DNA.

T

Fig. 1.1: Schematic representation of the Central Dogma

Source: http://thesciencecatalyst.blogspot.in/

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DNA Replication in Prokaryotes Page 7 of 28 A. DNA – From “Nuclein” to the Deoxy-ribonucleic Acid

Discovery: Nucleus became the focus of research on heredity with Ernst Haeckel (1866) postulating that hereditary information is transmitted from one generation to the next through nucleus. Friedrich Miescher, who performed extensive studies on the nucleus in WBCs obtained from pus, isolated the nuclear material from nucleus and named it ‘nuclein’ (1870-1871). He further identified nuclein as a distinct biomolecule and established that it was made up of nearly similar proportions of an acidic and a basic component and contained significant amount of phosphorus (Messer 2005; “Biochemistry, Genetics, and Replication of DNA” 2005, “Website”

2017a; James D. Watson 2011; Lagerkvist 1998). Nuclein kept changing names as and when further characterization studies continued.

The term ‘chromatin’ was coined by Walther Flemming (1879) (Gr. “colour”) for the nuclein present in the form of diffused mass of thin thread like substance which could be stained by certain chemical stains (Portugal and Cohen 1980; Gregory and Ryan Gregory 2007). Wilhelm Waldeyer (1888) observed the distinct rod shaped conspicuously staining form of condensed chromatin inside nucleus and used the term ‘chromosome’ (Gr. ‘colour body’). As studies to reveal the chemical nature of this biomolecule continued, Richard Altman (1889) conclusively proved the non- protein nature of nuclein. Based on the overall acidic nature of the biomolecule Altman is also known to have proposed the term “nucleic acid” which certainly was the more appropriate and accepted term for the substance (Gregory and Ryan Gregory 2007; “Website” 2017b). Much later in 1914, Robert Feulgen formulated a specific stain for nucleic acid and the Feulgen stain even till date happens to be one of most routinely used DNA stains for chromosomal analysis in cytological and histological experiments (Derenzini et al. 2000).

Phoebus Aaron Theodore Levene (1869 – 1940) is credited with a number of landmark discoveries about DNA chemistry. While working at the Rockefeller Institute of Medical Research, Levene identified the two forms of nucleic acid, both RNA and DNA and also identified and characterized all the components of DNA, including the four types of nitrogen bases, the phosphate group, the ribose sugar (1909), the phoshodiester linkage and most importantly the deoxyribose sugar (1929) (Hunter 1999; Bass 1940; Morange and Cobb 2000). However, inspite of all these significant contributions, one major stumbling block against establishing DNA as the genetic material was also posed by the erroneous ‘tetra-nucleotide theory’

proposed by Levene (1910) in which he stated that DNA is composed of simple monotonous repeats of the four constituent nucleotides (i.e., -ATCGATCG….-) and thus it was unlikely to be a genetic material as it lacked the diversity and flexibility essential for an informational macromolecule (Hunter 1999; “Website” 2017c; Allison 2011).

DNA v/s Protein: During the early decades of 20th century, sufficient evidences had surfaced in favor of complexity, variability and specificity of proteins. A long DNA v/s

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DNA Replication in Prokaryotes Page 8 of 28 protein debate ensued between supporters of the two groups, one favoring DNA as

the genetic material while the other favoring protein as the genetic material. There was an increasing notion DNA was merely a structural unit and protein was the key player as a functional and hereditary unit for the following reasons (Hausman 2015) (“Website” 2017d):

 Chromosomes were known to be made up of DNA and proteins

 DNA was known to be made up of only 4 different subunits, present as monotonous invariable repeats

 Chromosomes were known to contain less DNA than protein by weight

 Proteins were known to include 20 different subunits, thus having greater potential for variety of combinations

It was only with a series of independent landmark observations that followed, which provided mounting evidences in favor of DNA that the last few missing links between DNA and heredity finally fell into place. Eventually DNA was established as the universal genetic material for all living organisms beyond all doubts.

B. DNA as the Universal Genetic Material

Griffith’s Transformation Experiment: Frederick Griffith (1928) provided the first demonstration of bacterial transformation and proposed the concept of

‘transforming principle’ (Krebs et al. 2012). Griffith further postulated that the

‘Transforming Principle’ was the carrier of genetic information as it was able to convert one phenotype to another, [Panel-1] thus exhibiting an inherent behavior of genetic material. The only missing link then, was to determine the exact chemical nature of the transforming principle which could then establish the nature of the genetic material (Allison 2011; Krebs et al. 2013; Hayes 1966).

Avery, MacLeod and McCarty Experiment: Oswald Avery, Colin MacLeod, and MacIyn McCarty (1944) determined the exact chemical nature of ‘transforming principle’ [Panel-2] and provided the final conclusive evidence that DNA is the transformation material (Avery, MacLeod, and McCarty 2007; Mahadevan 2007)(Fry 2016a)

Hershey-Chase Experiment: Alfred Hershey and Martha Chase (1952) showed that DNA is also the genetic material at least in certain virus. Using the ‘Waring blender experiment’ with radiolabeled protein [³²S] or the DNA [³²P] incorporated T2 bacteriophage and its unlabeled host bacteria, Hershey and Chase provided another evidence that DNA is genetic material (Fry 2016b; Krebs et al. 2012)(“Website”

2017e).

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DNA Replication in Prokaryotes Page 9 of 28 Experiments with TMV: A. Gierer and G. Schramm (1956) while working with a

virus infecting tobacco plants discovered that this virus had RNA as its genetic material. Heinz Fraenkel-Conrat and B. singer (1957) devised the technique disintegrate the TMV particles and isolate RNA and protein from TMV, but they did not find any trace of DNA. These observations demonstrated for the first time that RNA could also act as the genetic material, in some virus (Gierer and Schramm 1956; Fraenkel-Conrat and Singer 1959; Creager 2002).

Point to Ponder – DNA v/s RNA: It is now known for sure that with the exception of only certain RNA virus, DNA is the universal genetic material for all known living organisms. But, there is a well-accepted hypothesis that RNA was the first molecule of heredity and it evolved all the essential methods for storing and expressing genetic information before DNA came into existence. Have you ever

Fig 1.2: Schematic representation of Griffith’s experiment demonstrating bacterial “transformation”

Source: https://sites.google.com/site/averymacleodmccarty/griffith-s-experiment

PANEL-1

As indicated in the following figure, in his classical genetics experiment with nonvirulent, rough (R) strain and virulent, smooth (S) strain of Streptococcus pneumoniae Griffith successfully showed that a ‘factor’ from the virulent strain could convert a non-virulent strain to a virulent strain. He further postulated that the ‘factor’ was the carrier of genetic information by which the non-pathogenic strain could be transformed (genetically changed) to pathogenic strain.

It was already accepted that an integral feature of the genetic material is to control phenotype. Thus, for any biomolecule to act as the genetic material, it must exhibit the ability to alter the phenotype of the recipient.

Griffith named this ‘factor’ as the ‘Transforming Principle’ as it was able to convert one phenotype to another, thus exhibiting an inherent behavior of genetic material. However, the exact chemical nature of the transforming principle was yet to be determined.

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wondered then, what could have been the possible factors and reasons which prompted the evolution and natural selection to favor DNA over RNA as the universal genetic material? Let’s try a simple exercise. Make a list of all the logical reasons you can think of, supporting DNA as the genetic material over RNA and when you.

Fig. 1.3: Schematic representation of AMM experiment demonstrating DNA is the ‘transforming principle’

Source:

https://sites.google.com/site/averymacleodm ccarty/amm-experiment

PANEL-2

Avery, MacLeod and McCarty took Griffith’s experiment one step ahead and performed a series of meticulously planned experiments to decipher the chemical nature of Griffith’s

‘transforming principle’. Instead of in vivo experiments in mice, they replicated Griffith’s experiment only with bacterial culture (Streptococcus pneumoniae cells expressed on blood agar) and studied the transformed bacterial phenotype. As shown in Fig 1.3, they extended the scope of their experiment as the ‘transforming material’ extracted from different sets of the heat-killed bacterial cells were individually treated with either protease or RNAse or DNAse. They observed that the only treatment which prevented transformation of the recipient bacterial culture was the DNAse treatment. Thus their experiments and observation conclusively demonstrated that the chemical nature of transforming principle was only DNA and not RNA or proteins which were the other two known constituents inside the nucleus.

Thus it was established that only pure DNA was the genetic material or heredity biomolecule.

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PANEL-3

It is well-accepted that RNA was the first molecule of heredity. It evolved all the essential methods for storing and expressing genetic information before DNA came onto the scene.

With progress in evolution, complicacies increased and different life forms evolved and eventually two-stranded DNA came into existence. Remnants of the precursor forms of hereditary molecule such as ssRNA or dsRNA or ssDNA do occur till date, but only in the non-cellular connecting link between living and non-living world, the virus!

However, evolution replaced RNA with DNA as the universal genetic material!!

Before we find out the reasons, let us first review the eligibility requirements for ‘genetic material’. A molecule supposed to function as genetic material is expected to have the following characteristic features:

 Replication: It should have the ability to replicate itself faithfully and completely before being transmitted to the offspring

 Stability: It should provide a stable storage for genetic information, that should have high resistance to damages and also an intrinsic damage repair strategy

 Evolution: It should have the ability to evolve and change itself to more efficient and diverse forms.

 Expression: It should be able to express the information efficiently in a closely regulated manner as and when needed.

Why Evolution Favored DNA?

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are done, check your list with Error! Not a valid bookmark self-reference. (Olby 2013)

C. DNA Structure: Journey to the Phenomenal Double-helix

Although DNA was already established as the genetic material of all living organisms, the structural and chemical properties of DNA was yet to be elucidated.

Two major obstacles in determining the correct structure were posed by the tetranucleotide theory of Levene and by the erroneous ‘triple helical’ structure predicted by Linus Pauling (Hunter 1999; J. Watson 2008; “Website” 2017e) . But as factual evidences kept falling into place with further investigations, the DNA research steadily progressed towards a phenomenal discovery.

Knowledge of the chemical structure of nucleotides had already previously established that each nucleotide was made up of a deoxyribose sugar, phosphate, and nitrogenous base. But their individual proportion and sequence in DNA was yet unclear. R. Hotchkiss (1948) and Chargaff (1949) analysed numerous DNA and RNA samples from various organisms put forth the first contradiction to

‘tetranucleotide theory’. By using basic techniques like paper chromatography and other analytical methods, they established that the four nucleotides were not necessarily present in unimolar proportions (Chargaff et al. 1949; Hotchkiss 1948) and thus suggested that the nucleotide sequence in DNA held the key to its biological importance and functional significance as a molecule of heredity.

Erwin Chargaff continued his research on the basis of the above hypothesis and performed base composition analysis of DNA by regulated hydrolysis and observed that the molar composition of the bases was different for DNA obtained from different sources and most importantly established an important numerical relationship known as the famous Chargaff’s Rule of Equivalence (1950). This rule which is universally valid till date states that the “molar concentration of adenine was identical to that of thymine and the molar concentration of guanine was identical to that of cytosine” (Chargaff and Davidson 1955; Hargittai 2005;

Chargaff 1963).

[A] : [T] = 1:1 [G] : [C] = 1:1

It was recognized that the difference in base composition of DNA obtained from various organisms reflected the basis of diversity in the living world and formed the basis of difference in the genetic information contained in this biomolecule of heredity.

One final step was to establish the molecular and geometrical organization of DNA. By combining the observations of X-Ray crystallographic studies of DNA conducted by William Astbury (1938) and the critical findings of X-Ray diffraction photography of DNA performed by Maurice Wilkins and Rosalind Franklin (1953) (Wilkins 2005; Johnson, Mertens, and Wilkins 1989) James Watson and

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Maurice Hugh Frederick Wilkins (Prize share: 1/3) James Dewey Watson

(Prize share: 1/3) Francis Harry Compton Crick

(Prize share: 1/3)

Francis Crick proposed the double helix model of DNA structure (1953) which is by far the universally established model for DNA structure. It states that the “DNA is a double stranded, regular, right handed helix with a diameter of 20 A and makes one complete turn every 34 A along the length of the molecule and each complete helix contains roughly ten nucleotide base pairs”. Watson and Crick also proposed the hypothesis of ‘semi-conservative’ nature of DNA replication (Krebs et al. 2012; Olby 2013; Portugal and Cohen 1980; James D. Watson 2011; J.

D. Watson and Crick 1953). Maurice Wilkins and Rosalind Franklin published their findings in the same issue of Nature (April, 1953) is which Watson and Crick published their findings.

Later in 1962, Francis Harry Compton Crick, James Dewey Watson and Maurice Hugh Frederick Wilkins (Fig. 1.4) shared the Nobel Prize in Physiology or Medicine

"for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material".

II. FUNDAMENTALS OF DNA REPLICATION

Towards the mid 1950’s, the structure, composition and chemical nature of the universal genetic material, DNA, were already documented. The one important gap to be filled up was to discover and establish the mode of replication and faithful propagation of the genetic material from the parental generation to the off springs.

Scientists kept hypothesizing different models of DNA replication until the exact nature of DNA replication, the ‘semiconservative’ nature, was established by the landmark genetics experiment of Meselson and Stahl (1957). Before going into the details of Meselson and Stahl’s experiment, let us have a quick look at the other models of DNA replication that were suggested and eventually ruled out (Fig. 2.1)

Fig. 1.4: Joint winners of the Nobel Prize in Physiology or Medicine 1962 Source: https://www.nobelprize.org/nobel_prizes/medicine/laureates/1962/

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Fig. 2.1: Classical models of DNA replication: A-Conservative; B-Dispersive; C-Semi-conservative.

Source: https://www.mun.ca/biology/scarr/iGen3_03-01.html

A B C

A. Suggested Models of DNA Replication

Conservative Replication Model: The proponents of this model suggested that during replication, the parental DNA unwinds and new daughter strands are synthesized based on parental strands but after the replication the two parental strands join back with each other. In other words, the original DNA molecule remains

‘conserved’ after replication. DNA replication thus results in synthesis of one molecule that consists of two new strands (with exactly the same sequences as the parent DNA molecule) and another molecule that consists of both original DNA strands (identical to the original DNA molecule) (Halazonetis 2014; Krebs et al.

2012).

Dispersive Replication Model: This pattern of DNA replications suggests that the parent DNA strands are broken into smaller segments which get randomly redistributed among the daughter DNA strands during replication. Thus the parent DNA gets fragments and “dispersed” among the fragments of new DNA during replication. At the end of replication, as per this model, synthesis of two DNA molecules occur, which are mixtures, or “hybrids,” of parental and daughter DNA. In this model, each individual strand is a patchwork of original and new DNA (Krebs et al. 2012; Araki 2016).

Semi-conservative Replication Model: The semi-conservative model of DNA replication proposes that during replication, as the two strands in the parent DNA unwind from each other, each strand then acts as the template for synthesis of a new, complementary strand. As the replication ends, each of the two new DNA molecules formed contain one original strand from the parent DNA (which is thus conserved) and one newly synthesized strand (Filner 1965; Araki 2016) (Filner 1965).

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Fig. 2.2: Schematic diagram showing the Meselson-Stahl experiment that established “semi- conservative” nature of DNA replication

Source: http://philschatz.com/biology-book/contents/m44487.html

B. Semi-Conservative Nature of DNA replication

This model of DNA replication first hypothesized by Watson and Crick (1953) happens to be the universally accepted model of DNA replication as it perfectly accounts for the structural and functional dynamics of DNA. Further this replication model alone can endorse DNA as the genetic material by explaining the mechanism of complete and faithful transmission of genetic information from the parent to the offspring generation.

Meselson and Stahl Experiment: Mathew Meselson and Franklin Stahl (1957) performed a classic genetics experiment by which by they successfully and definitively demonstrated the semi-conservative nature of DNA replication (Fig. 2.2) (Stillman 2002; Krebs et al. 2012). E. coli was grown for 14 generations in heavy nitrogen (¹⁵NH4Cl) containing medium and then shifted to lighter isotope (¹⁴NH4Cl) medium. Thereafter, DNA extracted from the E. coli after every generation was subjected to CsCl2 density gradient centrifugation and the resulting bands were analyzed. DNA molecules containing ¹⁵N being heavier than DNA containing ¹⁴N should form a distinct band at a lower position. They observed that the DNA extracted after first round of replication banded at intermediate position and with every subsequent cell division the band corresponding to ¹⁴N containing DNA kept increasing exponentially and the band corresponding to ¹⁵N containing DNA kept decreasing proportionally. This observation could be explained only if the DNA replication was semi-conservative in nature.

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It’s worth mentioning here, that Meselson and Stahl are also credited with inventing

“density-gradient centrifugation” technique for this experiment which today finds numerous other applications (Fry 2016c; Dasgupta and Boston University School 2013).

C. Characteristic Features of DNA Replication

The two integral properties of genetic material of organisms are to contain the genetic information for one generation and also to faithfully replicate and transmit the information across generations. DNA, which is the universal genetic material, has its structural and functional properties conserved across all life forms. The basic mechanism of DNA replication, including the important components involved in the process, are thus also conserved across all domains of life, be it prokaryotes or eukaryotes.

The basic mechanism of DNA replication exhibits certain conserved features across all living organisms, which are summarized below. There are, albeit, certain striking differences between DNA replication in prokaryotes and in eukaryotes which shall be discussed at the end of DNA replication in eukaryotes.

1) Semiconservative DNA Replication: This feature has been discussed in detail in the preceding section. It must be remembered that DNA replication in all known living organisms exhibits the semiconservative nature.

2) Semi-discontinuous DNA Replication: This mode of replication implies that the DNA synthesis does not occur uniformly on both strands; instead, one of the two new strands is synthesized in a seamless continuous manner, while the other strand is synthesized in a discontinuous manner by successive formation of multiple fragments of DNA (Wang 2005; DePamphilis 1996; Alberts et al. 2014).

DNA synthesis occurs by means of polymerization of deoxyribonucleotides, one at a time, thus forming long polynucleotide chains. This addition of nucleotides is achieved by complementary base-pairing directed by the nucleotide sequence in the template strand of parent DNA. It is also known that DNA polymerase (also known as DNA pol, DNA-dependent DNA polymerase enzyme) is the master enzyme and the key player for all known DNA replication and DNA polymerization events.

Further, it has been observed that all of the known primary DNA polymerases share two conserved features:

 DNA pols can polymerize DNA only in the 5’3’ direction, antiparallel to the template strand; and

 DNA pols lack de novo synthesis ability.

Thus, DNA synthesis by DNA polymerase requires a template present in 3’5’

orientation and also a primer whose 3’-OH position must be free and available to DNA polymerase for addition of the next complementary nucleotide. A series of

A B C

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events occur following the unwinding of DNA duplex leading to synthesis of new DNA strands on each DNA template strand, which can be summarized as follows:

 Priming: Synthesis of a short stretch of RNA (also in 5’  3’ direction) complementary to the template strand by Primase, a special type of DNA dependent RNA polymerase (with inherent de novo synthesis ability) resulting in formation of RNA/DNA heteroduplex

 Polymerase switching: DNA polymerase takes over and starts adding deoxyribonucleotides after the primer has been synthesized.

 Primer removal: RNA primer is later removed by 5’  3’ exonuclease action of DNA pol I enzyme which simultaneously incorporates DNA bases in place of RNA bases.

If you observe the diagram (Fig 2.3), you would understand that due to antiparallel orientation of DNA strands in the DNA duplex, at the replication fork, only one of the two template strands is available in the required 3’  5’ orientation (Leading strand) relative to the replication fork while the other template strand is present in reverse orientation (Lagging strand) (Wang 2005).

Consequently DNA synthesis

progresses in a continuous seamless manner only the Leading strand in overall 5’

 3’ direction which is along the direction of movement of replication fork. In case of the Lagging strand, however, although the new DNA strand appears to grow in 3’

 5’ direction, but the synthesis actually occurs discontinuously, in the form of multiple short stretches of DNA being synthesized in 5’  3’ direction (extending in opposite direction relative to replication fork). These short fragments of DNA are known as the Okazaki fragments (Okazaki 2017), each of each of which requires about 10 bases of RNA primer to begin synthesis (as discussed above). Hence, DNA synthesis on the lagging strand begins a short while after (lags behind) the beginning of DNA synthesis on the leading strand, when, due to progression of the replication fork, more than 10 bases on the lagging template strand are exposed and available for RNA priming and Okazaki fragment synthesis. Later the RNA primers are removed and Okazaki fragments are joined and covalently sealed by DNA ligase to form an intact lagging strand (Wang 2005; DePamphilis 1996; Alberts et al. 2014).

Both DNA strands, however, must be synthesized in concert by a dimeric DNA polymerase located at the replication fork. The lagging parental strand wraps in a trombone like structure to facilitate the process.

Fig. 2.3: Semi-discontinuous mode of DNA replication

Source:http://oregonstate.edu/instruct/bb350/textmaterials/10/Slide09.jpg

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Fig. 2.4: Formation of replication bubble and bidirectional movement of replication fork in (A) E. coli and (B) Eukaryotic chromosome.

Source: http://oregonstate.edu/instruct/bb350/textmaterials/ch10.html

A B

3) Bidirectional Replication of DNA: In the light of various experimental evidences available regarding direction of DNA replication (Alberts et al. 2014; Krebs et al.

2012) it is now well accepted that at the site of Origin of replication (discussed in a later section), a replication bubble is formed by the unwinding of the DNA duplex.

The replication bubble is flanked by one replication fork at each end and the two replication forks progress in opposite direction away from each other. Thus the replication bubble expands in both directions resulting in bidirectional replication of DNA (Fig. 2.4). DNA replication occurs simultaneously on both the DNA templates but the new strands extend in opposite directions relative to each other.

In case of prokaryotes, having a single circular chromosome with a single origin of replication, a single replicon is formed where a single replication bubble flanked by a pair of replication forks is formed. In eukaryotes where the chromosome is paired and linear in nature containing multiple origins of replication, multiple replicons are formed, one at each origin (Lodish et al. 2012; Alberts et al. 2014).

III. DNA REPLICATION IN PROKARYOTES

The DNA content in prokaryotes, as we already know, is distributed in two forms:

 The genomic or chromosomal DNA and

 The non-genomic, extrachromosomal plasmid DNA

Both, the chromosomal and the plasmid DNA are composed of a single closed circular DNA molecule present in coiled or supercoiled form. The plasmid and the chromosomal DNA do not share much similarity beyond this. The plasmid replicates independently and multiple times in between two successive cell divisions while the genomic DNA replicates only once between two cell divisions. In this module we shall focus only on the mechanism of replication of genomic DNA in prokaryotes and

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highlight the regulatory mechanism for the same. But before we get started with the replication steps and mechanism, let us have a quick look at the genome of prokaryotes.

Prokaryotic Genome: Genome is defined as the haploid set of chromosomes in a gamete or microorganism or in each cell of a multicellular organism which carries one complete set of genes or genetic material present in a cell or organism.

Prokaryotic genome is characterized by the following important features:

 It is composed of a single, circular closed chromosome

 The circular prokaryotic chromosome has a single point origin of replication, known as oriC and single point of termination characterized by ter sequences

 The chromosome in prokaryotes is present in the cytoplasm attached to the plasma membrane

 As the prokaryotes lack a conspicuous nucleus, the genome is not enclosed inside a nucleus

 The prokaryotic genome is largely devoid of introns

A. Enzymes Involved In Bacterial DNA Replication

Prokaryotes are arguably the simplest of organisms, but that does not make the DNA replication machinery of prokaryotes any simple. The basic mechanism of DNA replication in fact is known to be conserved across all domains of life. The discovery of E. coli DNA polymerase by the Arthur Kornberg in 1956 (who named it as DNA polymerase I) (“DNA Polymerase I (Pol I; Kornberg Enzyme, Kornberg Polymerase, DNA-Dependent DNA Polymerase I; EC 2.7.7.7)” 2015) paved the way for studying DNA replication machinery of living organisms and thereby developing strategies to manipulate the phenomenon.

Fig. 3.1: Genome map of E. coli indicating the important regions(A) and the gene sequence coding for enzymes involved in DNA replication (B)

Source: (A) http://www.nibb.ac.jp/annual_report/1997/22.html (B) https://goo.gl/images/Q6x3TJ

A B

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As of today nearly 30 conserved proteins and protein complexes are known to participate in prokaryotic DNA replication (T. Kornberg and Kornberg 1974; A.

Kornberg and Baker 2005; Brutlag et al. 1969; Mchenry and Kornberg 1981). We begin with a summary of important enzymes (Fig. 3.2) that participate, regulate and drive the replication of DNA in prokaryotes.

1) DNA Polymerase: the master enzyme of the replication machinery that is mainly responsible for polymerisation, editing and repair of DNA strands. Three different types of DNA polymerase are known in prokaryotes:

(i) DNA pol I – DNA pol I was the first DNA polymerase ever to be discovered and identified by Arthur Kornberg in E. coli lysate. This polymerase is a large 103 kDa protein, expressed in highest concentration in E. coli. Upon mild protease treatment DNA pol I dissociates into two fragments, a large 68 kDa Klenow fragment and a small 35 kDa fragment. The functional roles of DNA pol I are listed below:

 Klenow fragment exhibits 5’  3’ Polymerase and 3’  5’ Proofreading exonuclease activity. It is mainly associated with DNA repair and gap filling

 35 kDa fragment exhibits 5’  3’ Exonuclease activity that is involved with removal of RNA primer and replacing with newly synthesized DNA

(ii) DNA pol II– Exhibits 5’  3’ Polymerase and 3’  5’ Proofreading exonuclease activity and is mainly involved in DNA Repair function

(iii) DNA pol III– This is the main replicative polymerase used in E. coli chromosome replication. It is a multi-subunit protein complex with at least ten subunits (Fig. 3.3) associating to form the DNA pol III holoenzyme (Marians et al.

1998). Although the primary function of DNA pol III is to polymerize DNA in 5’  3’ direction, the different subunits are known to have different functions as tabulated below (Table 3.1)

Fig. 3.2: Assembly of enzymes at the replication fork in prokaryotes Source: http://philschatz.com/biology-book/contents/m44488.html

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Fig. 3.3: Schematic diagram of DNA plo III holoenzyme Source:https://en.wikipedia.org/wiki/DNA_polymerase_III_holoenzyme#/m

edia/File:DNA_polymerase_III_(with_subunits).jpg

Name of the subunit

Encoding

Gene Primary function

α subunit dnaE Catalytic subunit; 5’ → 3’ polymerase ε subunit dnaQ 3'→5' proofreading exonuclease activity θ subunit holE Stimulates the ε subunit's proofreading activity and

dimerization of Pol III β subunits dnaN Acts as the sliding DNA clamps,

τ subunits dnaX Dimerize the core enzymes (α, ε, and θ subunits) γ subunit dnaX Acts as the clamp loader for the lagging strand

2) Helicase: unwinds the DNA double helix by denaturing the hydrogen bonds between the nitrogen bases on the complementary strands (Soultanas 2005).

3) Primase: this enzyme is a special type of DNA dependent RNA polymerase with de novo synthesis ability that synthesizes RNA primers required for initiating DNA replication (Chamberlin 1974).

4) Sliding clamp: a multi subunit protein complex that holds the replisome in place and prevents dropping off of DNA pol as the replication fork progresses

5) SSB-protein: a single stranded DNA is unstable and fragile in nature. When the DNA duplex unwinds, single strand binding proteins bind on to the two single stranded DNA templates in order to stabilize the two strands and prevent rewinding of DNA duplex.

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6) Topoisomerase: unwinding of DNA duplex induces supercoiling and torsional stress in the unwound part of DNA just ahead of the replication fork. This stress is released by Topoisomerase-IV which relaxes the supercoils by inducing nicks in the DNA followed by resealing of the DNA backbone.

7) DNA ligase: As discussed in the preceding section, DNA synthesis on the lagging strand occurs in the form of discontinuous Okazaki fragments. After the removal of RNA primers and extension of Okazaki fragments, the single nucleotide gap between two adjacent Okazaki fragments is sealed off by DNA ligase.

8) Tus protein: Synthesized by the tus gene, the Terminus Utilization Substance (Tus) protein binds to the termination (ter) sequences on the E. coli chromosome.

The Ter-Tus complex mediates arrest of replication fork and termination of DNA replication (MacAllister, Khatri, and Bastia 1990; Oakley 2015).

B. Origin of Replication, Primosome Complex and Replisome Complex in Bacteria

OriC: Origin of replication refers to specific recognition sites on the chromosome where DNA replication begins. The origin of replication in E. coli also known as oriC is characterized by the following consensus sequences (Zyskind and Smith 1986):

 Three stretches of 13-mer AT-rich sequences(13 x3)

 Four stretches of AT-rich nanomer sequences (9 x 4)

Pre-replication Complex: The pre-replication complex shows high affinity for the oriC and binds to DNA selectively at the oriC sequences. This is followed by the unwinding of DNA duplex by DNA helicase and simultaneous assembly of all other enzymes and proteins (Primosome and Replisome complexes) essential for precise and complete replication of DNA. Pre-replication complex in bacteria is a multi- protein complex that binds to the oriC prior to unwinding of DNA and contains the following proteins (Zawilak-Pawlik, Nowaczyk, and Zakrzewska-Czerwińska 2017;

Jelena and Drag 2011; Gao 2016):

 Fis (Factor for Inversion Stimulation) – DNA bending protein

 Seq A

 Dna A – the main component that recognizes and binds tightly to a 9-base pair consensus sequence in oriC: 5' – TTATCCACA – 3

 IHF (Integration Host factor) – DNA bending protein

Primosome: Primosome refers to a large multi-protein complex that binds to the oriC at the time of initiation of DNA replication. All the proteins and enzymes essential to trigger DNA synthesis are closely associated to form the primosome which finally synthesizes RNA primer that acts as the precursor for DNA synthesis.

As we know, that DNA replication occurs in a semi-discontinuous manner, the

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primosome functions only once on the leading strand, but repeatedly on the lagging strand for synthesis of Okazaki fragments.

E. coli primosome (Fig. 3.4), a 600 kDa protein assembly (Jeiranian et al. 2013;

Tanaka and Masai 2010), includes seven main components whose functions are listed below:

 DnaG Primase: DNA dependent RNA polymerase; synthesizes RNA primers

 DnaB helicase: forms a complex with DnaC and causes unwinding of DNA duplex

 DnaC helicase assistant: associates with helicase

 PriA

 Pri B

 PriC

 DnaT: essential component of the primosome and required for chromosomal DNA replication; though whose exact function is yet unknown

 Dam methylase: methylates the adenine in the 5’-GATC-3’ sequence at oriC Priming: E.coli DNA pol-III lacks de novo synthesis ability and hence DNA synthesis by DNA pol-III on both, leading and lagging strands, requires RNA primer as a precursor. Priming is term for collective events leading to synthesis of the RNA primer and thereby forming the RNA/DNA chimeric duplex. This is the primary function of the enzyme Primase (DnaG), the DNA-dependent RNA polymerase present in primosome complex.

Fig. 3.4: Assembly of bacterial primosome and replisome at oriC prior to DNA replication Source: http://reasonandscience.heavenforum.org/t1849p15-dna-replication-of-prokaryotes

form a sub complex that initially binds to the DNA and triggers recruitment of other primosome complex proteins

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Replisome: At the end of priming, the RNA primers are excised by the 3’  5’

exonuclease activity of DNA pol I, which also fills the gap by incorporating deoxy- riboneucleotides. The immediate subsequent steps involve movement of the replication fork along the direction of replication which is achieved by a large macromolecular multi-protein complex called the replisome. Replisome in E. coli facilitates movement of the replication fork and addition of nucleotides to the nascent DNA strands at a remarkable rate of 1000 nucleotides per second. It includes all the proteins and enzymes essential for replication of DNA and elongation of the nascent DNA strands on both leading and lagging templates. Major components of replisome are known to be conserved across all domains of life; the components of E. coli replisome are listed below (Nina Y. Yao and O’Donnell 2010; N. Y. Yao and O’Donnell 2016):

 Dimeric complex of DNA pol III (holoenzyme) – Main replicative polymerase

 DNA pol I – Primer removal and Gap filling by dNTPs

 β/ γ/ τ complex – Clamp protein and clamp loader complex

 SSB protein – Binds to and stabilizes single stranded DNA

 DnaB Helicase – Unwinding the DNA duplex

 DnaG Primase – RNA priming

 Gyrase (Topoisomerase) – Relaxes stress and facilitates unwinding of DNA

 DNA ligase – Seals gap between Okazaki fragments

Replication of DNA faces two main constraints that DNA pol III can polymerize only

in the 5’  3’ direction and that a dimeric complex of DNA pol III, which is a part of the same replisome, synthesizes nascent strands on both the leading and lagging templates in a concerted manner. Consequently the lagging strand must turn back to access the replisome forming a trombone like structure (Fig. 3.5). It is also a function of the replisome to accommodate for the loop like structure of the lagging strand and stabilize its interaction with DNA pol III.

Fig. 3.5: Schematic representation of arrangement of replisome and looping back of lagging strand to be coupled with the leading strand

Source: http://reasonandscience.heavenforum.org/t1849-dna-replication-of-prokaryotes

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C. Steps Involved in DNA Replication in Prokaryotes

Replication of DNA in prokaryotes involves an elaborate, complex yet highly coordinated series of events from the time of assembly of preRC to the segregation of a pair of daughter chromosomes. The complete sequence of events may be grouped into three segments, namely, Initiation, Elongation and Termination of DNA replication.

Initiation: Initiation of DNA replication is triggered by assembly of pre-RC at oriC, leading to recruitment of primosome complex followed by unwinding of DNA duplex (Fig. 3.6). This is immediately followed by “RNA priming” by Primase and formation of the dimeric RNA/DNA hetero-duplex. This step is critical because the DNA polymerase can add a nucleotide only to the free 3’-OH position of an available nucleotide. Biochemical reaction mediated by DNA 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 in-coming dNTP, complementary to the next base in the template strand.

This is accompanied by elimination of the pyrophosphate. Polymerization of DNA is a high energy consuming reaction which derives energy from hydrolysis of dNTPs and the resulting pyrophosphates (Alberts et al. 2014; A. Kornberg and Baker 2005;

Krebs et al. 2012)

When a short stretch of RNA primer (~10 bases) has been synthesized, DNA pol I begins adding DNA bases and extending the RNA primer till the replisome has assembled. Functional switching from Primase to DNA pol I followed by recruitment of replisome complex marks the end of initiation and beginning of elongation phase.

Elongation: This phase involves synthesis of nascent DNA strands on the leading as well as lagging template strand. The event of priming is immediately followed by helicase mediated unwinding of DNA duplex, formation of the replication bubble and simultaneous release of torsional stress in the DNA duplex by Gyrase, which acts as a Topoisomerase (Runyon and Lohman 1993; Liu and Wang 1978). A dimeric complex of DNA pol III which is an integral part of the replisome complex then continues polymerization of DNA on both leading and lagging strands in a concerted

Fig. 3.6: Initiation of DNA replication in E. coli

Source: http://www.nature.com/nrmicro/journal/v11/n5/fig_tab/nrmicro2994_F1.html

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but semi-discontinuous manner. The leading strand is replicated seamlessly but the lagging strand is replicated in the form of multiple short segments of DNA known as Okazaki fragments (Fig. 3.7). A primosome is required for synthesis of each Okazaki fragment on the lagging strand (Okazaki 2017).

Replication bubble is flanked by one replication fork stabilized by its own replisome on either side. Bidirectional progression of replication fork is facilitated by the respective replisomes extending in opposite direction thus expanding the replication bubble from oriC to terminal sequence. Okazaki fragments are elongated by DNA pol I and sealed by DNA ligase to form the intact lagging strand. DNA pol III- subunits polymerize DNA while DNA pol III- subunits do the proofreading (Wu et al. 1992). As discussed earlier, SSB and Gyrase form a part of the replisome complex. During the entire course of events, the unwound single strands of DNA are stabilized by the single strand binding proteins which coat the DNA around the replication fork to prevent rewinding of the DNA. Topoisomerase (Gyrase) binds to the DNA at the region ahead of the replication fork to prevent supercoiling and facilitate smooth progression of replication fork.

Termination: Replication E. coli chromosome terminates when the replication bubble expanding on either side of the origin meet at a position on the chromosome approximately opposite to the position of oriC. The site of termination is characterized by two clusters of ter elements (TerA-TerG) (Fig. 3.8) which are 23 base pair consensus DNA sequences that provide the binding site for the Tus protein.

Fig. 3.7: Elongation of replication fork in E. coli

Source: http://www.nature.com/nrmicro/journal/v11/n5/fig_tab/nrmicro2994_F1.html

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Replication fork progressing bi-directionally is trapped by the Ter-Tus complex marking the termination of DNA replication. After the completion of replication, the two daughter DNA duplexes which are still interlinked are unlinked by the double strand break action of Topoisomerase II (Oakley 2015; MacAllister, Khatri, and Bastia 1990).

IV. FIDELITY OF DNA REPLICATION IN PROKARYOTES

DNA replication is a pivotal process for survival and proliferation of all organisms.

Although DNA replication is a highly complex sequence of multiple events, yet it is precisely regulated and coordinated to ensure high precision or fidelity. Faithful and complete replication of DNA prior to cell division is ensured by at least three serial fidelity steps (Fig. 4.1) during chromosomal replication in E. coli, which collectively provide a remarkable escape rate as low as 10⁻⁹ to 10⁻¹¹ errors per base pair per round of replication (Fijalkowska, Schaaper, and Jonczyk 2012; Alberts et al. 2014;

Lodish et al. 2012).

1) Base Selection: this fidelity check step utilizes the inherent property of DNA polymerase to discriminate against inserting an incorrect base (error rate~10⁻⁵)

2) 3'5' Exonuclease Action: 3'→5' proofreading exonuclease activity of the Klenow fragment and ε subunit of DNA pol III holoenzyme facilitate editing

A B

Fig. 3.8: (A) Termination of DNA replication and (B) Terminus region in E. coli Source: http://reasonandscience.heavenforum.org/t1849p25-dna-replication-of-prokaryotes

Fig. 4.1: General scheme indicating three serial fidelity steps during chromosomal replication

Source:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3391330 /pdf/nihms-364222.pdf

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of mis-inserted bases by DNA polymerase (escape rate ~10⁻²)

3) Strand-directed Mismatch Repair: This step involves removal of remaining mismatches by post-replicative DNA Mismatch Repair (MMR) (escape rate

~10⁻³)

V. SUMMARY