This free living protozoa reproduces by asexual and sexual methods. In Paremoecium, the asexual reproduction is caused by transverse binary fission.

Binary Fission: Just before division the animal ceases feeding, the central portion of the body bulges. Micronucleus increases in size and fine spindle fibres are formed. Chromosomes divide eumitotically passing through all the stages of mitosis and each part goes to opposite pole. The macronucleus divides amitotically. One part of each of macronucleus and the micronucleus with the chromosome set go to the anterior part while the other goes to the posterior part. By this time a furrow appears at the centre at right angle to the orientation of the spindle. This furrow deepens into sharp constrictions which cut the animal into two in the transverse plane.

The oral groove or cytopharynx is inherited by the daughter at the anterior end. Regeneration of lost parts takes place in both of them. Two paramoecia develop from one parent by transverse binary fission.

Asexual reproduction in Paramoecium depends on-

(i)   Temperature of water.

(ii) Quality and quantity of food.

Significance of binary fission

All the daughter paramoecia produced from the same parent have the same genetical constitution and so they form a clone.

Systematic Position of Paramoecium

Sub-Kingdom- Protozoa
Phylum- Ciliophora
Genus- Paramoecium
Species- caudatum

Structure of Paramoecium

Locomotion in Paramoecium

As paramecium is a ciliate animal so it moves by its cilia. The rapid swimming is facilitated by the beating of fine and hair-like cellular organelles, called cilia, that cover the animal’s entire cell-body. Paramoecium moves with a speed of 1500 µm or more per second.

Structure of Cilia under light microscope-

Cilia are the principal locomotory organs of Paramoecium. Structurally, cilia and flagella are similar but they differ in their arrangements and number. Flagella are one, two or a few having localized arrangement but cilia are arranged in rows. Flagella are longer but cilia are shorter. In Paramoecium cilia are arranged in longitudinal rows. The entire body is covered with cilia so it is known as Holotrichus.

Structure of Cilia under light microscope-

The microfibrils forming the basic skeleton of a cilium together known as Kinetodesmata. Each kinetodesma resides within the cytoplasm and directly areises from a basal granule Cilia are arranged in a row and each row is known as kinety. Each cilium shows a complicated arrangement of basal granule and kinetodesma. In a kinetodesma the number of fibrils are may exceed 500. Besides these there are contractile microfibrils below the pellicle of Paramoecium. They may join to basal granules.

On the basis of the structure each cilium of a Paramoecium is divided into two regions. They are-

(i)                  Kinetodesmal region and (ii) Shaft region.

Each kinetodesmal region shows (9+2) fibrillar arrangement like a flagellum, i.e., there are nine peripheral and fibrils and two central fibrils. Each cilium originates by a double root, roots join to basal granules from which kinetodesmata arise. Each cilium is bounded by a unit membrane and it is continuous with the plasma membrane. Each outer fibril consists of two sub-fibrils, sub-fibril A and sub-fibril B. The central two fibrils have no sub-fibrils and remain bounded by a common sheath. According to Gibbons (1967) the sheath of the central fibrils gives out nine radially arranged spoke like structure which joins with the sub-fibril A. The inner arm of subfibril A joins with the sub-fibril B of the next fibril in a clock-wise direction. The basal bodies, kinetodesmata and tubules of the cilia together constitute the infraciliature of ciliated animal like Paramoecium.

Process of ciliary locomotion

Like flagellate protozoa ciliate protozoa also live in water and therefore, their movements are caused by striking the water with cilia. These strokes are irregular and contain only one wave. Each cilium has an effective stroke and a recovery stroke. In the recovery stroke there is no movement. As cilia are many but shorter in length so there is a coordination in their movement and this coordinated movement is known as metachronal movement.

During an effective stroke the protein molecules of the central fibrils contract and the cilia bend to strike the water. This is the effective stroke which creates sufficient force to drive the animal forward. After the effective stroke is completed cilia tend to return to its original position generating minimum force by alternate events of that helped in effective stroke. This is recovery stroke. Effective strokes and recovery strokes are the main principles of ciliary locomotion. Some cilia of the kinety perform effective stroke whereas some recovery stroke. In this way a wave of metachronal rhythm pass from anterior to posterior region and the animal tend to move forward. As the oral cilia are larger and they beat more quickly thus helping the animal to rotate on its axis and the animal moves forward while rotating on its own axis.

Molecular mechanism of Ciliary Movement-

To explain the mechanism of ciliary movement at the molecular level two theories have been proposed. They are-

1. The localized contraction theory- According to this theory contractile units are placed at regular intervals along the length of the axoneme. The contraction of these units help to bend the cilia. During ciliary movement  there is a change in the length of the subfibrils of a doublet. Molecular contraction and relaxation would explain both the effective and recovery stroke. During effective stroke one set undergoes contraction while other set streatches and during the recovery stroke the reverse would take place.
2. The sliding filament model- This theory states that bending of the cilium is initiated by sliding of the tubules of the peripheral fibrils relative to one another. The two subfibres A and B of a doublet do not move relative to one another because they share a common wall although the termination distance of the subfibres remain constant during the effective and recovery strokes. So, it is supposed that the filaments must be moving against adjacent doublet and thus effects a ciliary movement.

Reproduction in Euglena

In E. viridis no sexual reproduction occurs. It reproduces by asexual binary and multiple fissions and undergoes encystment. Under favorable conditions euglenas reproduce by longitudinal binary fission.  The longitudinal binary fission is always symmetrogenic, that is, the parental Euglena divides into two daughter individuals, where one is the plane mirror image of the other.

A. Binary Fission:

1. The nucleus divides into two mitotically followed by division of cytoplasm. In prophase stage all the nuclei (endosomes) fuse together and each chromosome splits into two daughter chromosomes or chromatids.  In metaphase all the chromatids are arranged in the equator. No spindle is formed at the anaphase, but the chromatids are separated and moved towards the opposite poles.  In telophase due to the constriction of the nuclear membrane the nucleus is finally separated into two daughter nuclei. Following the nuclear division all the anterior extra-nuclear organelles such as the blepharoplasts, reservoir, cytophraynx and stigma, etc. are all duplicated.
2. The division of nucleus (karyokinesis) is immediately followed by division of cytoplasm (cytokinesis) where due to the longitudinal splitting of the cytoplasm the Euglena divides into two daughter euglenae.
3. In some cases the stigma breaks into component granules. Ordinarily the original flagellum of the parent is retained by one daughter euglena and the other develops a new one. On the other hand, some observers have reported complete disappearance of the entire locomotory apparatus during division, and each daughter cell reconstructs a new set.

B. Multiple Fission:

Cases of multiple fission, though rare, have also been reported. Multiple fission takes place in encysted condition. Euglena very readily encysts forming both thick and thin walled cysts within which it divides into several (16-32) daughter englenas. Sometimes the flagellate loses its flagellum and rounds up into an alga-like cell in which metabolisms continues and reproduction occurs by fission, thus, forming extensive green scums on the surface of ponds. In this condition, they are said to assume the Palmella state. Such a palmella stage is of regular occurrence in some species.

Encystment:

Under certain conditions ordinary protective cysts are also formed. Encystment is stimulated by lack of food, lack of oxygen, drying, heat (as in strongly illuminated cultures) and fouling of the medium. The cyst is composed of a special carbohydrate and is of yellowish brown colour. The cysts are generally rounded, their walls being made of two or three concentric layers. The cysts are usually small, their total width being equal to the diameter of the animal, it may be larger sometimes. Thin and stalked cysts have also been reported in some species and in others each may be provided with an operculum. The organisms may lie in the centre of the cyst or towards one side (excentrally). The cysts are protective structures that help the organisms to withstand unfavorable circumstances and also help their dispersal. On the return of favorable conditions the cysts dissolve and the organisms come out and begin normal life.

Warner and Satir (1974) described the structure of a flagellum as seen under Electron Microscope. According to them various microtubules extending from the base to the tip of the flagellum construct the basic frame work of a flagellum. These are altogether eleven microtubules of which nine are situated at the periphery and two at the centre forming a (9+2) arrangements. All the fibrils are enclosed within a protoplasmic sheath continuous with the cell membrane. The nine peripheral fibrils and two central fibrils constitute the axonome.

Each peripheral tubule is composed of two sub-fibrils namely subfibrils namely subfibril A and subfirbil B. Sub fibrils A and B are also connected by very short arms. All the microfibrils of axoneme are composed of tubulin dimmer. Each dimer is again formed of two monomers namely ? and ? monomers. Each subfibril of the centre is composed of thirteen protofilaments. The central two fibrils are covered by a membrane and the peripheral fibrils are connected with the membrane through radially arranged spoke-like arms. The energy required for flagellar movement comes from ATP.

The following classification recommended by the Society of Protozoologists (Levine et.al., 1980), the subkingdom Protozoa is divided into seven phyla:

1) Sarcomastigophora, 2) Apicomplexa, 3) Myxozoa, 4) Microspora, 5) Labyrinthomorpha, 6) Ascetospora & 7) Ciliophora.

1) Phylum Sarcomastigophora;

1. Single type of nucleus, except in Foraminiferida.
2. Sexuality, when present, essentially syngamy.
3. With flagella, pseudopodia or both type of locomotor organelles.

i) Subphylum Mastigophora:

1. One or more flagella typically present in trophozoites.
2. Asexual reproduction basically by intrakinetal (symmetrogenic) binary fission.
3. Sexual reproduction known in some groups.

Example- Giardia, Trichomonas etc.

ii) Subphylum Sarcodina:

1. Pseudopodia, or locomotive protoplasmic flow without discrete pseudopodia.
2. Flagella, when present, usually restricted to developmental or other temporary stages.
3. Body naked or with external or internal test or skeleton.
4. Asexual reproduction by fission.
5. Sexuality, if present, associated with flagellate or, more rarely, amoeboid gametes.
6. Most species free-living.

Example- Entamoeba, Acanthamoeba, Naegleria etc.

iii) Subphylum Opalinata:

1. Numerous cilia in oblique rows over entire body surface.
2. Cytostome absent.
3. Nuclear division accentric.
4. Binary fission generally interkinetal.
5. Known life cycles involve syngamy with anisogamous flagellated gametes.
6. All parasitic.

Example- Opalina, Zelleriella etc.

2) Phylum Apicomplexa:

1. Apical complex (visible with electron microscope), generally consisting of polar ring(s), rhoptries, micronemes, conoid, and subpellicular microtubules present at some stage.
2. Micropore(s) generally present at some stage.
3. Cilia absent.
4. Sexuality by syngamy.
5. All species parasitic.

Example- Monocystis, Gregarina, Toxoplasma, Plasmodium, Babesia etc.

3) Phylum Myxozoa:

1. Spores of multicellular origin, with one or more polar capsules and sporoplasms.
2. With one, two, or three (rarely more) valves.
3. All species parasitic.

Example- Myxidium, Ceratomyxa, Trilospora etc.

4) Phylum Microspora:

1. Unicellular spores, each with imperforate wall, containing one uninucleate or dinucleate sporoplasm and simple or complex extrusion apparatus always with polar tube and polar cap.
2. Without mitochondria.
3. Often, if not usually, dimorphic in sporulation sequence.
4. Obligatory intracellular parasites in nearly all major animal groups.

Example- Burkea, Hessea, Glugea etc.

5) Phylum Labyrinthomorpha:

1. Trophic stage, ectoplasmic network with spindle-shaped or spherical, non-amoeboid cells.
2. In some genera amoeboid cells move within network by gliding.
3. Zoospores produced by most species.
4. Saprobic or parasitic on algae, mostly in marine and estuarine water.

6) Phylum Ascetospora:

1. Spore apparently multicellular (or unicellular?).
2. With one or more sporoplasms.
3. Without polar capsules or polar filaments.
4. All parasitic.

Example-  Urosporidium, Haplosporidium etc.

7) Phylum Ciliophora:

1. Simple cilia or compound ciliary organelles typical in at least one stage of life cycle.
2. With subpellicular infraciliature present even when surface cilia absent.
3. Two types of nuclei, with rare exception.
4. Binary fission transverse, but budding and multiple fission also occurs.
5. Sexuality involving conjugation, autogamy and cytogamy.
6. Nutrition heterotrophic.
7. Contractile vacuole typically present.
8. Most species free-living, but many commensal, some truly parasitic, and a large number found as phoronts on a variety of hosts.

Example-  Balantidium, Trichodina, Nyctotherus etc.

Introduction:

Amoeba is the most popular, free-living available protozoan. It is commonly found on the bottom mud or on underside of aquatic vegetation in freshwater ponds, ditches, lakes, springs etc. It moves and feeds with the help of false or pseudopodia, formed as a result of streaming flow of cytoplasm. Read the rest of this entry »

Membrane Transport Processes

1. Diffusion:  the migration of molecules or ions as a result of their own random movements, from a region of higher concentration to a region of lower concentration.
2. Osmosis: – is the movement of water through a semipermeable membrane movement of water (at constant temp. and pressure) is from the solution with lower  concentration of solutes to the solution of  higher concentration of solutes (or from the more pure water to less pure water)
3. Tonicity: Tonicity refers to the relative concentration of solute on either side of a membrane.

Isotonic

In an isotonic solution, the concentration of solute is the same on both sides of the membrane (inside the cell and outside). A cell placed in an isotonic solution neither gains or loses water. Most cells in the body are in an isotonic solution.

Hypotonic

A hypotonic solution is one that has less solute (more water). Cells in hypotonic solution tend to gain water.

Hypertonic solution

A hypertonic solution is one that has a high solute concentration. Cells in a hypertonic solution will lose water.

4.         Facilitated Transport/ Diffusion:

-  the transport of molecules across a cellular membrane thru specific protein channels / carrier molecule (facilitating pathway) from a region of high conc. to a region of low conc.

-  process is driven by conc. differences and does  not require energy.

5. Active Transport:

-  the pumping of molecules or ions across a cellular membrane through a carrier protein

-  from a region of lower concentration to one of higher concentration.

-  therefore against the “current” or concentration gradient.

-  such a process requires energy.

In cells, some molecules must be moved against their concentration gradient to increase their concentration inside or outside the cell. This process requires the input of energy and is known as active transport. As with facilitated diffusion, special transporters in the membrane are used to move the molecules across the membrane. The plasma membrane is not the only cellular membrane that requires active transport. All organelles surrounded by membranes must concentrate some molecules against their concentration gradients.

Active Transport appears to be of two general types, (i) primary active transport and (ii) secondary active transport.

(i) Primary active transport is directly related with chemical energy (ATP) or electric energy (electron flow). Exampless of primary active transport are Na+, K+ translocating ATPase in mammals and proton translocating ATPase of bacteria.

Types of Active Transporters

There are three types of active transporters in cells: (1) Coupled transporters link the “downhill” transport of one molecule to the “uphill” transport of a different molecule; (2) ATP -driven pumps use the energy stored in adenosine triphosphate (ATP) to move molecules across membranes; (3) Light-driven pumps use the energy from photons of light to move molecules across membranes. Light driven pumps are found mainly in certain types of bacterial cells.

Most of the energy expended by a cell in active transport is used to pump ions out of the cell across the plasma membrane. Because ions have an electrical charge, they do not easily cross membranes. This phenomenon allows large ion concentration differences to be built up across a membrane. Highly selective transporters are present in membranes that pump certain ions up their concentration gradients, but ignore other ions.

The NA ⁺ -K ⁺ Pump. One of the best understood active transport systems is the sodium-potassium pump, or NA ⁺ -K ⁺ pump. This carrier protein is a coupled transporter that moves sodium ions out of the cell while simultaneously moving potassium ions into the cell. Because of the pump, the sodium ion concentration inside the cell is about ten to thirty times lower than the concentration of sodium ions in the fluid surrounding the cell. The concentration of potassium ions inside the cell is almost exactly the opposite, with a ten-to thirtyfold higher concentration of potassium ions inside the cell than outside.

Because the cell is pumping sodium from a region of lower concentration (inside) to a region of higher concentration (outside), the NA ⁺ -K ⁺ pump must use energy to carry out its pumping activity, and this energy is supplied by ATP. For this reason, the NA ⁺ -K ⁺ pump is also considered an enzyme . It belongs to a class of enzymes known as ATPases that use the energy stored in ATP to carry out another action. Other membrane transporters use the energy from ATP to pump ions like calcium, amino acids , and other electrically charged molecules either into or out of the cell.

Ions carry a positive or negative electrical charge so that these gradients have two components: a concentration gradient and a voltage or electrical gradient. For instance, sodium ions are positively charged. The higher concentration of sodium ions outside of the cell than inside means that outside of the cell will have a positive charge and the inside of the cell will have a negative charge. This potential difference, or voltage, across the membrane can be used as an energy source to move other charged molecules. Positively charged molecules will be attracted towards the inside of the cell and negatively charged molecules will be attracted to the outside of the cell. It is, in fact, this electrical potential that causes positively charged potassium ions to enter the cell through the Na-K pump, even though they are moving up their concentration gradient.

The potential energy of the gradient can be used to produce ATP or to transport other molecules across membranes. One of the most important uses of the NA ⁺ gradient is to power the transport of glucose into the cell. The NA ⁺ -glucose cotransporter moves sodium down its concentration gradient, and glucose up its gradient, as both move into the cell.

(b) Secondary active transport- It depends upon chemiosmotic energy (membrane potential or and/or ion gradients). Examples of secondary active transport are the glucose transport system of the intestinal epithelium of mammals and the lactose permease system in E.coli.

The free surface of the intestinal epithelium has numerous microvilli which are formed by projections of the brush border membrane. Primary active transport results in the Na+ being pumped in the cell. The electrochemical sodium ion gradient can be then utilized for secondary active transport of glucose in to the cell against the concentration gradient. Thus there is glucose-Na+ co-transport catalyzed by a glucose carrier. Such sodium-dependent transport has been observed for various amino acids and sugars in different vertebrates and for amino acids in bacteria.

The sodium pump maintains a higher concentration of Na+ outside the cell than on the inner side. This results in a tendency for Na+ to enter the cell. This is done in the form of a carrier-sugar or carrier-amino acid complex.

INTRODUCTION:

DNA replication, the basis for biological inheritance, is a fundamental process occurring in all living organisms to copy their DNA. This process is “replication” in that each strand of the original double-stranded DNA molecule serves as template for the reproduction of the complementary strand. Hence, following DNA replication, two identical DNA molecules have been produced from a single double-stranded DNA molecule. Cellular proofreading and error checking mechanisms ensure near perfect fidelity for DNA replication.

In a cell, DNA replication begins at specific locations in the genome, called “origins”. Unwinding of DNA at the origin, and synthesis of new strands, forms a replication fork. In addition to DNA polymerase, the enzyme that synthesizes the new DNA by adding nucleotides matched to the template strand, a number of other proteins are associated with the fork and assist in the initiation and continuation of DNA synthesis.

DNA structure:

DNA is a long polymer made from repeating units called nucleotides.

In living organisms, DNA does not usually exist as a single molecule, but instead as a pair of molecules that are held tightly together.

These two long strands represent themselves in the shape of a double helix. The nucleotide repeats contain both the segment of the backbone of the molecule, which holds the chain together, and a base, which interacts with the other DNA strand in the helix. A base linked to a sugar is called a nucleoside and a base linked to a sugar

and one or more phosphate groups is called a nucleotide. If multiple nucleotides are linked together, as in DNA, this polymer is called a polynucleotide.

The backbone of the DNA strand is made from alternating phosphate and sugar residues. The sugar in DNA is 2-deoxyribose, a pentose (five-carbon) sugar. The sugars are joined together by phosphate groups that form phosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings. These asymmetric bonds mean a strand of DNA has a direction. In a double helix the direction of the nucleotides in one strand is opposite to their direction in the other strand: the strands are antiparallel. The asymmetric ends of DNA strands are called the 5? (five prime) and 3? (three prime) ends, with the 5′ end having a terminal phosphate group and the 3′ end a terminal hydroxyl group. This directionality has consequences in DNA synthesis, because DNA polymerase can only synthesize DNA in one direction by adding nucleotides to the 3′ end of a DNA strand.

One major difference between DNA and RNA is the sugar, with the 2-deoxyribose in DNA being replaced by the alternative pentose sugar ribose in RNA.

The DNA double helix is stabilized by hydrogen bonds between the bases attached to the two strands. The four bases found in DNA are adenine (abbreviated A), cytosine (C), guanine (G) and thymine (T). These four bases are attached to the sugar/phosphate to form the complete nucleotide.

DNA Replication Begins at Specific Chromosomal Sites

Most important decision every cell has to make is whether, and when, to replicate its DNA. DNA replication is controlled at the initiation step. Control would be most efficient if there are specific sites on chromosomes at which DNA replication always begins in vivo.

Molecular studies indicate that replication of these DNAs actually begins at a defined sequence of base pairs near the center of these bubbles, called the replication origin .

Replication origin - Stretch of DNA that is necessary and sufficient for replication of a circular DNA molecule, usually a plasmid or virus, in an appropriate host cell.

E. coli Replication Origin

The E. coli replication origin oriC is an ?240-bp DNA segment present at the start site for replication of E. coli chromosomal DNA.

oriC contain repetitive 9-bp and AT-rich 13-bp sequences, referred to as 9-mers (dnaA boxes) and 13-mers, respectively, these are binding sites for the DnaA protein that initiates replication.

Three Common Features of Replication Origins

1. Replication origins are unique DNA segments that contain multiple short repeated sequences.

2. These short repeat units are recognized by multimeric origin-binding proteins. These proteins play a key role in assembling DNA polymerases and other replication enzymes at the sites where replication begins.

3. Origin regions usually contain an AT-rich stretch. This property facilitates unwinding of duplex DNA because less energy is required to melt A·T base pairs than G·C base pairs. Origin-binding proteins control the initiation of DNA replication by directing assembly of the replication machinery to specific sites on the DNA chromosome.

Genes
dnaA, dnaB, dnaC, dnaG

Proteins
DnaA, DnaB or Helicase, DnaC, Primase, SSB

DnaA Protein Initiates Replication in E. coli

Genetic studies first suggested that initiation of replication at oriC most likely depended on the protein encoded by a gene designated dnaA. Subsequent genetic studies with recombinant E. coli further pinpointed the DnaA protein as a prime candidate for interaction with oriC. In vitro studies showed that pure DnaA protein binds to the four 9-mers in oriC, forming an initial complex that contains 10-20 protein subunits.

Binding of DnaA to the oriC 9-mers facilitates the initial strand separation, or “melting,” of E. coli duplex DNA, which occurs at the oriC 13-mers. This process requires ATP and yields a so-called open complex.

The 9-mers and 13-mers are the repetitive sequences

• Multiple copies of DnaA protein bind to the 9-mers at the origin and then “melt” (separate the strands of) the 13-mer segments.
• The role function of DnaC is to deliver DnaB, which is composed of six identical subunits, to the template.
• One DnaB hexamer clamps around each single strand of DNA at oriC, forming the prepriming complex. DnaB is a helicase, and the two molecules then proceed to unwind the DNA in opposite directions away from the origin.

DnaB Is an E. coli Helicase that Melts Duplex DNA

Further melting of the two strands of the E. coli chromosome to generate unpaired template strands is mediated by the protein product of the dnaB locus, a helicase that is essential for DNA replication. One molecule of DnaB, a hexamer of identical subunits, clamps around each of the two single strands in the open complex formed between DnaA and oriC. This binding requires ATP and the protein product encoded by the dnaC locus, which “escorts” DnaB to the DnaA proteins, yielding the prepriming complex.

Helicases constitute a class of enzymes that can move along a DNA duplex utilizing the energy of ATP hydrolysis to separate the strands. In E. coli, the separated strands are inhibited from subsequently recoiling by a single-strand-binding protein (SSB protein), which binds to both separated strands.

Helicases like DnaB bind to a single-stranded segment of DNA, then move along that strand melting the hydrogen bonds that link it to its complementary strand. Like many proteins that bind to DNA, helicases exhibit a directionality with respect to the unwinding reaction.

DnaB moves along the single strand of DNA to which it binds in the direction of its free 3 end, and in this sense it is said to unwind DNA in the 5 ‘- 3′ direction.

E. coli Primase Catalyzes Formation of RNA Primers for DNA Synthesis

As noted earlier, DNA polymerases can only elongate existing primer strands of DNA or RNA. The primers used during DNA replication in both prokaryotes and eukaryotes are short RNA molecules whose synthesis is catalyzed by the RNA polymerase primase. Primase is usually recruited to a segment of single-stranded DNA by first binding to a DnaB hexamer already attached at that site. The term primosome is now generally used to denote a complex between primase and helicase, sometimes with other accessory proteins. In initiation of E. coli DNA replication, a primosome is formed by binding of primases to DnaB in the prepriming complex. After the bound primases synthesize short primer RNAs complementary to both strands of duplex DNA, they dissociate from the single-stranded template.

At a growing fork, one strand is synthesized from multiple primers

(a) The overall structure of a growing fork.

Synthesis of the leading strand, catalyzed by DNA polymerase III, occurs by sequential addition of deoxyribonucleotides in the same direction as movement of the growing fork.

(b) Steps in the discontinuous synthesis of the lagging strand.

This process requires multiple primers, two DNA polymerases, and ligase, which joins the 3 – hydroxyl end of one (Okazaki) fragment to the 5 -phosphate end of the adjacent fragment.

(c) DNA ligation. During this reaction, ligase transiently attaches to the 5 phosphate of one DNA strand, thus activating the phosphate group.

The Leading and Lagging Strands Are Synthesized Concurrently

Once the prepriming complex and an RNA primer are formed at the E. coli replication origin, chain elongation to yield the leading strand proceeds with little difficulty. As we’ve seen, however, lagging-strand synthesis proceeds discontinuously from multiple primers. Two molecules of core DNA polymerase III are bound at each growing fork; one adds nucleotides to the leading strand, and the other adds nucleotides to the lagging strand. Coordination between elongation of the leading and lagging strand is essential; otherwise one template strand would be incorporated into a duplex with a newly synthesized complementary strand while large parts of the other template strand would remain single-stranded.

1. A single DnaB helicase moves along the lagging-strand template toward its 3 end, thereby melting the duplex DNA at the fork.

2. One core polymerase (core 1) quickly adds nucleotides to the 3 end of the leading strand as its single-stranded template is uncovered by the helicase action of DnaB. This leading-strand polymerase, together with its b -subunit clamp, remains bound to the DNA, synthesizing the leading strand continuously.

3. A second core polymerase (core 2) synthesizes the lagging strand discontinuously as an Okazaki fragment. The two core polymerase molecules are linked via a dimeric t protein.

4. As each segment of the single-stranded template for the lagging strand is uncovered, it becomes coated with SSB protein and forms a loop. Once synthesis of an Okazaki fragment is completed, the lagging-strand polymerase dissociates from the DNA but the core remains bound to the t -subunit dimer. The released core polymerase subsequently rebinds with the assistance of another b clamp in the region of the primer for the next Okazaki fragment.

E. coli DNA Polymerase III Catalyzes Nucleotide Addition at the Growing Fork

Three DNA polymerases (I, II, and III) have been purified from E. coli. In addition to its role in filling the gaps between Okazaki fragments, DNA polymerase I probably is the most important enzyme for gap filling during DNA repair. DNA polymerase II functions in the inducible SOS response discussed later; this polymerase also fills gaps and appears to facilitate DNA synthesis directed by damaged  templates.  Our discussion here focuses on DNA polymerase III, which catalyzes chain elongation at the growing fork in E. coli.

Termination of replication

Because bacteria have circular chromosomes, termination of replication occurs when the two replication forks meet each other on the opposite end of the parental chromosome. E coli regulate this process through the use of termination sequences which, when bound by the Tus protein, enable only one direction of replication fork to pass through. As a result, the replication forks are constrained to always meet within the termination region of the chromosome.

Eukaryotes initiate DNA replication at multiple points in the chromosome, so replication forks meet and terminate at many points in the chromosome; these are not known to be regulated in any particular manner. Because eukaryotes have linear chromosomes, DNA replication often fails to synthesize to the very end of the chromosomes (telomeres), resulting in telomere shortening. This is a normal process in somatic cells — cells are only able to divide a certain number of times before the DNA loss prevents further division. (This is known as the Hayflick limit.) Within the germ cell line, which passes DNA to the next generation, telomerase extends the repetitive sequences of the telomere region to prevent degradation. Telomerase can become mistakenly active in somatic cells, sometimes leading to cancer formation.

Summary of DNA replication

A self-correcting DNA polymerase catalyzes nucleotide polymerization in a 5′-to-3′ direction, copying a DNA template. Since the two strands of a DNA double helix are antiparallel, this 5′-to-3′ DNA synthesis can take place continuously on only one of the strands at a replication fork (the leading strand). On the lagging strand short DNA fragments are made by a “backstitching” process. Because the self-correcting DNA polymerase cannot start a new chain, these lagging-strand DNA fragments are primed by short RNA primer molecules that are subsequently erased and replaced with DNA.

DNA replication requires the cooperation of many proteins, including (1) DNA polymerase and DNA primase to catalyze nucleoside triphosphate polymerization, (2) DNA helicases and single-strand binding proteins to help open up the DNA helix so that it can be copied, (3) DNA ligase and an enzyme that degrades RNA primers to seal together the discontinuously synthesized lagging-strand DNA fragments, (4) DNA topoisomerases to help relieve helical winding and tangling problems, and (5) initiator proteins that bind to specific DNA sequences at a replication origin and catalyze the formation of a replication fork at that site.

At a replication origin a specialized protein-DNA structure is formed that subsequently loads a DNA helicase onto the DNA template; other proteins are then added to form the multienzyme “replication machine” that catalyzes DNA synthesis.

Enzymes of Dna Replication

Helicases: Helicases are proteins that unwind DNA using energy of ATP and create two potential replication forks.

SSB protein (single-stranded DNA–binding protein): SSB proteins prevent reanealing of single stranded DNA.

DNA topoisomerases:

Topoisomerase I: DNA molecules can coil and bend in space, leading to changes in topology, including formation of negative or positive supercoils. Local unwinding of a DNA duplex whose ends are fixed causes stress that is relieved by supercoiling. Any enzyme that cleaves only one strand of a DNA duplex and then reseals it is classified as a type I topoisomerase (Topo I). The Topo I from E. coli acts on negative, but not positive, supercoils.

Topoisomerase II or DNA gyrase:

Topo II enzymes have the ability to cut both strands of a double-stranded DNA molecule, pass another portion of the duplex through the cut, and reseal the cut in a process that utilizes ATP.

DNA ligase: It catalyzes formation of a phosphodiester bond between a 3’ hydroxyl at end of one DNA strand and a 5’ phosphate at end of another strand.

Primase: Primase synthesizes a short RNA primer, which remains base-paired to template DNA that is extended at 3’ end by DNA polymerase.

DNA polymerases: DNA polymerases are main polymerizing enzymes that add deoxyribonucleotides to 5’ end of RNA primer.

The proto-oncogene can become an oncogene by a relatively small modification of its original
function. There are three basic activation types:
*A mutation within a proto-oncogene can cause a change in the protein structure, causing
- an increase in protein (enzyme) activity
- a loss of regulation
* An increase in protein concentration, caused by
- an increase of protein expression (through misregulation)
- an increase of protein (mRNA) stability, prolonging its existence and thus its activity in the
cell
- a gene duplication (one type of chromosome abnormality), resulting in an increased amount
of protein in the cell
*A chromosomal translocation (another type of chromosome abnormality), causing
- an increased gene expression in the wrong cell type or at wrong times
- the expression of a constitutively active hybrid protein. This type of aberration in a dividing
stem cell in the bone marrow leads to adult leukemia

Mutations in microRNAs can lead to activation of oncogenes. New research indicates that small RNAs 21-25 nucleotides in length called microRNAs (miRNAs) can control expression of these genes by downregulating them. Antisense messenger RNAs could theoretically be used to block the effects of oncogenes.

A proto-oncogene is a normal gene that can become an oncogene due to mutations or increased expression. Proto-oncogenes code for proteins that help to regulate cell growth and differentiation. Proto-oncogenes are often involved in signal transduction and execution of mitogenic signals, usually through their protein products. Upon activation, a proto-oncogene (or its product) becomes a tumor-inducing agent, an oncogene. Examples of proto-oncogenes include RAS, WNT, MYC, ERK, and TRK.