Consequences of Complement Activation – Effector Functions

The MAC and other complement by-products formed during activation of various pathways augments the immune response in many ways as follows. These are known as effector functions of complement products.

1. Cell lysis by MAC

MAC damages cell membrane by making pores or channels in it and allowing the free passage of various ions and water in to the cell. This ultimately leads to cell death. Bacteria, enveloped viruses, irreversibly damaged cells, cancerous cells etc are killed by this mechanism commonly referred to as complement mediated cell lysis.

Gram positive bacteria, which are protected by their thick peptidoglycan layer, bacteria with a capsule or slime layer around their cell wall, or non-enveloped viruses are less susceptible to lysis.

2. Inflammatory Response

C3a, C4a and C5a are called anaphylotoxins. They bind to receptors on mast cells and induce its degranulation leaing to release of histamine and other pharmacologically active mediators of inflammation. They cause vasoconstriction and increase in vascular permeability. Along with the C5b67, they also induce the migration of neutrophils and monocytes to the site of complement activation. This leads to an inflammatory response which is a local protective response.

3. Opsonization

C3b and C4b act as major opsonins that coat the immune complexes and particulate antigen. Phagotytic cells express receptors (CR1, CR3 and CR4) for complement components (C3b, C4b etc.) and are able to bind the complement coated antigen and enhance its phagocytosis. C5a augments this process by enhancing the CR1 expression on phagocytes by 10 folds.

4. Viral Neutralization.

Complement plays a very important role in viral neutralization in many ways:

  • Most viral particles bind to their serum antibody and form particulate immune complex which stimulates the classical pathway. Many viruses are also capable of activating the alternative and lactin pathway in the absence of antibody. So, the MAC is able to destroy the viruses.
  • C3b helps in the formation of viral aggregates by acting as opsonin and thus decrease the net number of infective viral particles. This effect is enhanced in the presence of serum antibody.
  • Complement products also coat the viral particles. This coating neutralizes the viral infectivity by blocking its attachment to target cell and enhancing its phagocytosis by macrophages through complement.
  • Complement cytolyse most enveloped viruses causing fragmentation and disintegration.

5. Solublization of Immune Complex

C3b plays an important role in removing immune complex from the blood. its binding to complexes facilitates their binding to CR1 on RBCs. Though CR1 are present in higher number is granulocytes than the RBCs, but because of much larger number of RBCs than granulocytes in blood, RBCs account for the 90% of the total CR1 in blood. Immune complexes bound to the RBCs are taken to liver and spleen where they are phagocytoed after seperation from RBCs.

Pathways of Complement Activation

The complement activation can be divided into three pathways, Classical, Lectin (mannose binding protein) and Alternative, all of which result in the activation of C5 and lead the formation of the membrane attack complex (MAC).

Stages of Complement Activation

There are three main stages in the activation of complement cascade by any pathway:

  • Formation of C3 Convertase
  • Formation of C5 Convertase
  • Formation of MAC Complex

The three pathways differ in the way C5 is broken down but after that the formation of MAC is essentially the same.

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1. Classical Pathway

The classical pathway is triggered primarily by immune complexes (containing antigen and IgG or IgM) in the presence of complement components 1, 4, 2, 3, Ca++ and Mg++ cations. IgG and IgM are most potent activators of this pathway. Since they need Antibody interaction to initiate the pathway its an antibody dependent pathway.

They react in the order C1q, C1r, C1s,C4, C2, C3, C5, C6, C7, C8 and C9. Different IgG subclasses have different potency to activate the pathway. While IgG1, IgG2 and IgG3 (most effective) can activate complement, IgG4 is not able to activate at all.

This pathway begins by the formation and activation of C1qr2s2 (C1 Macro molecular complex) by the antigen-antibody complex and finally ends in the formation of membrane attack complex (MAC).

Initiation:

  • C1 is the first complement component to participate in classical pathway. It act as initiation and recognition unit. It is a macro molecular complex composed of three polypeptide chains – C1q, C1r and C1s. The over all molecule appears like a bunch of six tulips. Each globular head has a binding site for Fc fragment of the antibody. Two molecules each of C1r and C1s are bounded to C1q in space between the heads and stems. The overall molecular structure of C1 complex is C1qr2s2.
  • The C1r and C1s are serine protease enzymes which are activated by C1q. C1r2s2 complex exist in an elongated S form when in free state. But when bound to C1q, it assume a different conformation resembling to figure 8.
  • The activation of classical pathway begins by binding of at least two globular heads of the C1qr2s2 complex with the antigen antibody complex through the Fc fragment of the antibody.
  • C1qrs can also bind to a number of agents including some retroviruses, mycoplasma, poly-inosinic acid and aggregated IgG, and initiate the classical pathway.

Formation of C3 Convertase:

  • Binding of Cqr2s2 globular heads leads to a conformational change in the stems of C1q.This change is sequentially transmitted to C1r2 and then to C1s2.
  • During this change, C1r is activated by auto cleavage. The altered C1r cleaves C1s. At this stage, Cqr2s2 complex is fully activated.
  • This cleaved C1s is capable of cleaving both C4 and C2. Activated C1s enzymatically cleaves C4 into C4a and C4b. C4b binds to the Ag-bearing particle or cell membrane while C4a remains a biologically active peptide at the reaction site.
  • C4b binds C2, which becomes susceptible to C1s and is cleaved into C2a and C2b. C2a remains complexed with C4b whereas C2b is released. C4b2a complex is known as C3 convertase.

Formation of C5 Convertase:

  • C3 convertase, in the presence of Mg++,cleaves C3 into C3a and C3b. C3b binds to the membrane to form C4b2a3b complex whereas C3a remains in the microenvironment. C4b2a3b complex functions as C5 convertase, which cleaves C5 into C5a and C5b. Generation of C5 convertase marks the end of the classical pathway. C5b initiates the formation of membrane attack complex.

Formation of Membrane Attack Complex (MAC):

  • The C3b components of C5 convertase binds C5 and split into C5a and C5b fragments. The smaller fragment diffuses away and the large fragments C5b binds to C5 convertase to form a C4b2a3b5b complex. This act as the catalyst to form the membrane attack complex.
  • The C5b is extremely labile unless stabilized by the binding to C6 and immobilized by binding to cell membrane. it therefore, binds sequentially to C6 and C7 to form the complex C5b67. This binding exposes a hydrophobic region on C7 through which it is able to penetrate the cell membrane.
  • This membrane bound C5b67 act as a receptor for C8 which thus binds to it. C8 also inserts itself into the membrane.
  • This membrane inserted complex, C5b678 has a catalytic property to bind C9 molecule and polymerize it into a 12-15 unit tubular channel of about 10nm diameter . This structure penetrate into the cell membrane and form channels or pores into the cell membrane. It allows the free passage of water into the cell leading to cell swelling and lysis.
  • Since the C5b6789 complex destroys the cell by attacking cell membranes, it is called membrane attack complex (MAC). This form of cytolysis is referred to as complement mediated cytotoxicity.

 

2.  Alternative Pathway:

  • Alternate pathway is so called because it bypasses the requirement o f antigen-antibody complex, C1, C2 and C4 components and thus it is part of the innate immune system.
  • It begins with the spontaneous activation of C3 in serum and requires three other serum proteins called Factors B, Factor D and properdin, all present in normal serum.
  • C3 is spontaneously broken in serum to C3a and C3b.
  •  The cleaved C3b binds to foreign antigens or to host cells. In the normal cell surface sialic acid residues destroy the bound C3b rapidly and so do not allow the activation of this pathway further. Because of the low concentration of sialic acid levels in foreign antigen surfaces, the bound C3b remains active for a longer period.
  • The bound C3b on the foreign antigen surface binds another protein, factor B, which is cleaved by Factor D to produce C3bBb (now C3 convertase).
  • This has very short life of about 5 minutes unless it is stabilized by properdin which increases the half life to 30 minutes. Hence the alternative pathway is also known as the properdin pathway.
  • Stabilized C3 convertase cleaves more C3 and produces C3bBb3b complex (C5 convertase), which cleaves C5 into C5a and C5b. C5b initiates the formation of membrane attack complex. The sequence of events for the formation of MAC is common in all the pathways.
  • The alternative pathway provides a means of non-specific resistance against infection without the participation of antibodies and hence provides a first line of defence against a number of infectious agents.
  • This pathway is initiated without the antibody by various cell surface constituents that are foreign to the host such as those of bacteria, virus, fungi, yeast, parasites and even virally infected or cancerous host cells. Many non pathogenic cells and molecules such as Human antibodies-IgG, IgA and IgE, Cobra venom factor (CVF) and other proteins (e.g. proteases, clotting pathway products) also can activate the alternative pathway.

 

3. The Lactin Pathway

  • C4 activation can be achieved without antibody and C1 participation via the lectin pathway. Three proteins initiate this pathway namely a mannan-binding lectin/protein (MBL), and two mannan-binding lectin-associated serine proteases (MASP and MASP2), all present in normal serum.
  • MBL binds to certain mannose residues on many bacteria and subsequently interacts with MASP and MASP2. The MBL-MASP-MASP2 complex is similar to Ab-C1qrs complex (of classical pathway) and leads to activation of C4, C2 and C3. The rest follows as in classical pathway.

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Complement System

The term “complement” was coined by Paul Ehrlich to describe the activity in serum, which could “complement” the ability of specific antibody to cause lysis of bacteria. Complement historically refers to fresh serum capable of lysing antibody-coated cells.

Complement system is composed of over 20 different proteins produced by hepatocytes,macrophages and intestinal epithelial cells. Fibroblasts and intestinal epithelial cells make C1, while the liver makes C3, C6, and C9. They are present in the circulation as inactive molecules.

Though some components are resistant to heat, heating serum at 56oC for 30 minutes destroys complement’s activity and thus they are not able to kill the microbes or pathogens present in the blood. Thus complement system are heat labile.  Serum complement levels, especially C3, often drop during infection as complement is activated faster than it is produced.

Several complement proteins are zymogens (proenzymes). Which means they are an inactive substance which is converted into an enzyme when activated by another enzyme. They get activated when infected with pathogen. When activated, they become proteases that cut peptide bonds in other complement proteins to activate them by the process called proteolysis. Proteolysis removes an inhibitory fragment exposing an active site. Complement proteins work in a cascade, where the binding of one protein promotes the binding of the next protein in the cascade.

The compliment components are named in various ways e.g. by numerals (C1- C9), by alphabetical symbols (factor D) and by common trivial names (homologus restriction factor). Complement components named by numerals are numbered in the order in which they were discovered. During activation, some complement components are cleaved into two unequal fragments. The larger part of the molecule is designated “b” while the smaller fragment “a”.

Small fragment,known as Anaphylotoxins, may diffuse away into the micro-environment to produce other effects (they act as chemotactic agents) while in most cases it is the “b” fragment that participates in the cascade reaction sequence or in other words binds to the surface of the cell to be lysed (the fragments of C2 are an exception to this rule: C2a binds to the membrane while C2b is freed into serum or tissue spaces).

Inactivated fragments are indicated by a small “i”. Enzymatically active forms are symbolized by a bar over the letter or number.

Activation of complement results in the production of several biologically active molecules such as MAC, which contribute to nonspecific immunity and inflammation. Complement is not antigen-specific and it is activated immediately in the presence of pathogen, so it is considered part of innate immunity. Since antibody also activates some complement proteins, complement activation is also part of humoral immunity. Their activation proceeds via different pathways in a cascade fashion leading to lysis.

A polymeric membrane attack complex (MAC) is formed following the activation of complement system. MACs breakdown the cell membrane leading to “pore” formation. Movement of ions and fluid into cell through these pores leads to cell lysis. Complement products opsonize the antigen which is then easily phagocytosed. Immune complexes also require complement products for their removal from the body. Also byproducts of complements, cascade induce inflammation which again augments the overall cellular response in the affected area. Beside all these, complements play important role in viral neutralization in several ways.

Complement proteins can be quantified directly by ELISA, and complement activity can be measured by the complement fixation test.

DNA Damage

Mutations arise not only from the errors in the replication but also from damage to the DNA. Some damage is caused by environmental factors, such as radiation and chemicals like mutagens, which are chemical agents that increase the rate of mutation. DNA also undergoes spontaneous damage from the action of water.

Mechanisms which causes DNA Damage include:

  1. Hydrolysis (Deamination and Depurination)
  2. Alkylation
  3. Oxidation
  4. Radiation Ractions
  5. Base analogue and intercalating agents.

1. Hydrolysis

The most frequent and important kind of hydrolytic damage is deamination of the base cytosin. Deamination is the removal of an amine group from a molecule. Enzymes that catalyse this reaction are called deaminases.

  • The deamination of cytosin to uracil happens at a significant rate in cells. Deamination can be repaired by a specific repair process which detects uracil, not normally present in DNA; otherwise the U will cause A to be inserted opposite it and cause a C:G to T:A transition when the DNA is replicated.
  • Deamination converts adenine to hypoxanthine, which hydrogen bonds to cytosine rather than to thymine; guanine is converted in to xanthine, which continues to pair with cytosin, though with only two hydrogen bonds.
  • Deamination of 5-methylcytosine to thymine also occur. 5- Methylcytosine occurs in the human genome at the sequence 5’CpG3′, which normally avoided in the coding regions of genes. if the meC is deaminated to T, there is no repair system which can recognise and remove it (becase T is normal base in DNA). This means that whatever CpG occurs in genes it is a “hotspot” for mutation.

Deamination of Bases

DNA also undergoes depurination by spontaneous hydrolysis of the N-glycosyl linkage, and this produces an abasic site (that is deoxyribose lacking a base) in the DNA.

Depurination in DNA is a chemical reaction of purine deoxyribonucleosides, deoxyadenosine and deoxyguanosine, in which the β-N-glycosidic bond is hydrolytically cleaved releasing a nucleic base, adenine or guanine

depurination

2. Alkylation

Alkylation is the transfer of an alkyl group from one molecule to another. In alkylation, methyl orethyl groups are transferred to reactive sites on the bases and to phosphates in DNA backbone.

Alkylations do not lead immediately to mispairing. However, they do make the bond between sugar and base more labile, or more apt to break. When this break occurs, it leaves an apurinic site, a sugar without its purine. This obviously cannot be replicated properly unless it is first repaired, but cells sometimes attempt to replicate apurinic DNA anyway. If they do, they frequently insert the wrong base across from an apurinic site, and this generates a mutation. Alkylation can also enhance the tendency of a base to form the rare (wrong) tautomer, which changes the base-pairing properties, leading to mutations. Moreover, all of the nitrogen and oxygen atoms involved in base-pairing are also subject to alkylation, which can directly disrupt base-pairing and lead to mutation.

fig2

Many environmental carcinogens, or cancer-causing agents, are electrophiles that act by attacking DNA and alkylating it. Many of the favorite mutagens used in the laboratory for the express purpose of creating mutations are also alkylating agents. One example is ethylmethane sulfonate (EMS), which transfers ethyl (CH3-CH2) groups to DNA. The product of this methylation, O6-rthylgaunine, often mispaires with thymine, resulting in the change of G:C base pair into an A:T base pair when the damaged DNA is repliacted.

3. Oxidation

DNA oxidation is the process of oxidative damage on Deoxyribonucleic Acid. It occurs most readily at guanine residues due to the high oxidation potential of this base relative to cytosine, thymine, and adenine. Reactive oxygen species (O2-, H2O2, and OH) are generated by ionizing radiation and by chemicals agents that generate free radicals. An important oxidation product is 8-hydroxyguanine, which mispairs with adenine, resulting in G:C to T:A transversions.

fig3

4. Radiation Ractions

Ultraviolet radiation (UV radiation) cross-links adjacent pyrimidines on the same DNA strand, forming pyrimidine dimers, usually thymine dimers. Figure shows the structure of a thymine dimer and illustrates how it interrupts base-pairing between the two DNA strands. These dimers block DNA replication because the replication machinery cannot tell which bases to insert opposite the dimer. As we will see, replication sometimes proceeds anyway, and bases are inserted at random. If these are the wrong bases, a mutation results.

fig3

Thymine dimers. (a) Ultraviolet light cross-links the two thymine bases on the top strand. This distorts the DNA so that these two bases no longer pair with their adenine partners. (b) The two bonds joining the two thymines form a four-membered cyclobutane ring (red).

Ultraviolet radiation has great biological significance; it is present in sunlight, so most forms of life are exposed to it to some extent. The mutagenicity of UV radiation explains why sunlight can cause skin cancer: Its UV component damages the DNA in skin cells, which leads to mutations that sometimes cause those cells to lose control over their division.

Damage by Gamma and X -rays:

The much more energetic gamma rays and X rays, like ultraviolet rays, can interact directly with the DNA molecule. However, they cause most of their damage by ionizing the molecules, especially water, surrounding the DNA. This forms free radicals, chemical substances with an unpaired electron. These free radicals, especially those containing oxygen, are extremely reactive, and they immediately attack neighboring molecules. When such a free radical attacks a DNA molecule, it can change a base, but it frequently causes a single- or double-stranded break. Single-stranded breaks are ordinarily not serious because they are easily repaired, just by rejoining the ends of the severed strand, but double-stranded breaks are very difficult to repair properly, so they frequently cause a lasting mutation. Because ionizing radiation can break chromosomes, it is referred to not only as a mutagen, or mutation-causing substance, but also as a clastogen, which means “breaker.”

5. Base analogue and intercalating agents.

Mutations ae al;so caused by compounds that substitue for normal bases (base analogs) or slip between the bases (intercalating agents) to cause errors in replication.

Base analogs are structurally similar to proper bases but differ in ways that make them treacherous to the cell. Thus base analogs are similar enough to the proper base to get taken by cells, converted in to nucleoside triphosphates, and incoporated into DNA during replication. But, because of the structural difference between these analogues and the proper bases, the analogues base-pair inaccurately, leading to frequent mistakes during the replication process.

one of the most mutagenic base analogs is 5-bromouracil, an analog of thymine. The presence of bromo substituent allows the base to mispair with guanine via enol tautomer.

5-bromouracil (5-BU) ia a derivative of uracil and behaves as a thymine analog, which increases the probability of a tautomeric shift from the normal keto form to the enol form, mis-pairing with guanine instead of adenine. After one round of replication, an A - T to G - C transition mutation results.

5-bromouracil (5-BU) ia a derivative of uracil and behaves as a thymine analog, which increases the probability of a tautomeric shift from the normal keto form to the enol form, mis-pairing with guanine instead of adenine. After one round of replication, an A – T to G – C transition mutation results.

Intercalating agents are flat molecules containing several polycyclic rings that bind to the equally flat purine or pyrimidine bases of DNA. Intercalating agents are compounds, such as proflavin, acridine and ethidium,  that can bind to the major and minor grooves of DNA and cause addition or deletion of bases during replication. They may result in a frameshift mutation, which can alter the codon reading frame and result in aberrant DNA transcription and replication.

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The Replicon Model of Replication Initiation

The initial replication fork requires the separation of the two strands of the DNA duplex to provide a template for the synthesis of both the RNA primer and new DNA. Specific genomic DNA sequence direct the initiation of DNA Replication. The specific sites at which DNA unwinding and initiation of replication occur are called Origin of Replication.

The Replicon Model of Replication Initiation:

In 963 Frabcois Jacob, Sydney Brenner and Jacques Cuzin proposed a model to explain the events controlling the initiation of replication in bacteria. It proposed two components that control the initiation of replication :

  • Replicator: Replicator is defined as the entire set of cis-acting DNA sequences that is sufficient to direct the initiation of Replication.
  • Initiator: Initiator is the protein which specifically recognizes a DNA elements in the replicator and activates the initiation of replication. The initiator protein is the only sequence specific DNA-binding protein involved in the initiation of replication.

The model proposed that all the DNA replicated from a particular origin as a replicon. In prokaryotes since there is only one origin of replication at the single chromosome, the entire chromosome is a single replicon. In contrast the presence of multiple origins of replication divides each eukaryotic chromosomes into multiple replicons.

Replicator:

The origin of replication is the site on DNA where the DNA is unwound and DNA synthesis initiates. Although the origin of replication is always part of the replicator, sometimes the origin of replication is only a fraction of the replicator.

DNA Sequences of Replicator Share two common features:

  • First, they include a binding site for the initiator protein.
  • They include a stretch of AT-rich DNA that unwinds readily but not spontaneously. Unwinding of DNA at replicators is controlled by the replication Initiation Proteins.

The single replicator required for E-Coli chromosomal replication is called oriC. There are two repeated motif that are critical for oriC function. The 9-mer motif is the binding site for the E-Coli initiator, DnaA, and is repeated four times at oriC. The 13-mer motif, repeated 3 times, is the initial site of ssDNA formation during initiation.

Initiator:

Initiator proteins typically perform 3 different functions:

  1. Binding to the replicator – Bind a specific sequence DNA sequence within the replicator.
  2. Unwind DNA – once bound to the DNA, they frequently distort or unwind a region of DNA adjacent to the site of binding.
  3. Recruiting other replication proteins – Initiator proteins interact with additional factors required for replication initiation, thus recruiting them to the replicator.

In Prokaryotes, for example, the E-coli initiator, DnaA binds the repeated 9-mer elements in oriC and is regulated by ATP. When bound to ATP, DnaA also interacts with DNA in the region of the repeated 13-mer repeats of oriC. These additional interactions result in the separation of the DNA strands over more than 20bp within the 13-mer repeat region. this unwound DNA provides an ssDNA template for additional replication proteins such ad DNA helicase to begin the RNA and DNA synthesis steps of replication.

In Eukaryotes, the initiator is known as the origin recognition complex (ORC). It is a complex formed of 6 proteins. Like DnaA in E-Coli, ORC binds and hydrolyze ATP. ATP is required for sequence-specific DNA binding at the origin. Unlike DNA binding ORC at the replicator does not separate the DNA strands. ORC is , however, required to recruit all the remaining replication proteins to the replicator before it unwinds. Unwinding the strands requires the origin to get activated. Origin activation occurs only when the cell enters S phase of cell cycle.

Unlike prokaryotic initiator, ORC of eukaryotes have two functions:

  1. Binding to the replicator
  2. Recruiting other replication proteins

once the initiator binds to the replicator, the remaining steps in the initiation of replication is largely driven by the protein-protein interactions and protein-DNA interactions that are sequence independent. The end result is the assembly of two replication fork.

Mismatch Repair System

The replication machinery achieves remarkably high degree of accuracy using a proofreading mechanism. Proofreading improves the fidelity of DNA replication by a factor about 100 . The proof reading exonuclease is not, however, foolproof. Some mis incorporated nucleotides escape detection and become a mismatch between the newly synthesized strand and the template strand.

If the misincoporated nucleotide in the first round of replication is not subsequently detected and replaced, the sequence change will become permanent in the genome in the second round of replication.  At this point the mismatch will no longer exist; instead it will have resulted in a permanent change in the DNA sequence.

Fortunately, a mechanism exists for detecting the mismatches and repairing them, known as Mismatch Repair System. This system increases the accuracy of DNA synthesis by an additional two to three orders of magnitude.

Aims of Mismatch repair system are as follows:

  • Quick response : It must scan the genome for mismatches. Because mismatches are transient (they are eliminated in the second of replication) the mismatch repair system must find and repair mismatches.
  • Accuracy: The system must correct the mismatch accurately. that is it must replace the misincoporated nucleotides in the newly synthesized strand and not the correct nuceotide in the parent strand.

Mismatch Repair Pathway in Prokaryotes:

  1. In E-Coli mismatches are detected by a dimer of the mismatch repair protein MutS. MutS scan the DNA, recognizing the mismatches from the distortion they cause in the DNA backbone.
  2. MutS embraces the the mismatch-containing DNA , inducing a kink in the DNA and a conformational change in MutS itself.
  3. This complex of MutS and mismatch containing DNA recruits MutL, a second component of mismatch repair system .
  4. MutL, in turn, activates the mutH, an enzyme that causes incision or nick on one strand near the site of the mismatch.
  5. Nicking is followed by the action of specific helicase and one of three exonucleases. The helicase unwind the DNA, starting from the incision and moving in the direction of the site of the mismatch, and exonucleases progressively digest the displaced single strand, extending to and beyond the site of the mismatched nucleotide.
  6. This action produces a single-stranded gap, which is then filled by DNA polymerase III and sealed with DNA Ligase.

  • How does the mismatch repair system know which of the two mismatched nucleotides to replace?

The E-Coli enzyme Dam methylase methylates A residue on both strands of the sequence 5′-GATC-3′. The GATC sequence is widely distributed along entire genome , and all of these sites are methylated by the Dam methylase. When replication fork passes through DNA that is methylated at GATC sites on both strands , the resulting daughter DNA duplex will have hemimethylated (that is methylated on only parent strand). Thus for few minutes, until the Dam mathylase catches up and methylates the newly synthesized strand, daughter DNA duplex will be methylated only on the strand that served as the template. Thus the newly synthesized strand lacks methyl group and are so marked and hence can be recognized as the strand for repair.

The MutH proteins binds at such hemimethylated sites, but its endonuclease  activity is normally latent. Only when it is contacted by MutL and LutS located at a nearby mismatch does MutH become activated. Once activated, MutH selectively nicks the unmethylated strand.

Mismatch repair system in Eukaryotes:

Eukaryotes cells also repair mismatches and do so using homologs to MutS (Called MSH proteins for MutS homologs) and MutL (called MLH and PMS). Indeed, eukaryotes have multiple MutS-like proteins with different specificities.

Even though eukaryotic cells have MMR, mismatch repair systems, they lack MutH and E-coli’s clever trick of using hemimethylation to tag the parent strand.

Following are the detailed view how Eukaryotic Mismatch repair system works.

All eukaryotic organisms, including yeast, mouse and human, have MutS homologs (MSHs) and MutL homologs (MLHs). The eukaryotic MMR system has been well conserved during the evolutionary process. However, in contrast to MutS and MutL in bacteria, which function as homodimers, in eukaryotes MSHs and MLHs form heterodimers with multiple proteins. Five highly conserved MSHs (MSH2 to MSH6) are present in both yeast and mammals.

MSH1, which is present in mitochondria, exists only in yeast . MSH4 and MSH5 show reproductive tissue- specific expression, and null mutations of these genes do not confer mutator phenotypes because they are involved in meiotic recombination but not postreplication repair. Genetic and biochemical studies have indicated that MSH2 is required for all mismatch correction in nuclear DNA, whereas MSH3 and MSH6 are required for the repair of some distinct and overlapping types of mismatched DNA during replication. These three MutS homologs make two heterodimers: MutSa (MSH2⁄MSH6) and MutSb (MSH2⁄ MSH3). The former plays the major role in recognition of mismatched DNA in eukaryotic MMR. That is, MutSa functions in the repair of base–base mispairs as well as a range of insertion ⁄ deletion loop mispairs, whereas MutSb primarily functions in the repair of insertion ⁄ deletion loop mispairs.

MutL homologs in eukaryotic organisms were identified as genes whose amino acid sequences showed high similarity with prokaryotic MutL proteins, or whose mutation phenotypes were increased levels of postmeiotic segregation (PMS) that resulted from a failure to repair mismatches in meiotic recombination intermediates. There are four homologs of MutL in both yeast and mammals. In a genetic analysis, defects in MLH1 and PMS1 in yeast resulted in more severe mutator phenotypes, reminiscent of those of MSH2 and MSH6, than defects in the two other MutL homologs. Also, MLH1 interacted with the other three MutL homologs in a yeast two-hybrid analysis. Overall, yeast MLH1⁄PMS1 and mammalian MLH1⁄PMS2 heterodimers (each known as MutLa) play a major role in mutation avoidance, and the other two heterodimers of MutL homologs take part in the repair of specific classes of mismatches. The biochemical activities and structure of MutL homologs are closely related to those of prokaryotic MutL proteins, especially in the N-terminal domain. The X-ray crystallographic structure of the conserved N-terminal 40-kDa fragment of human PMS2 resembles that of the ATPase fragment of E. coli MutL.

Extensive genetic studies in yeast have failed to find orthologs of MutH and UvrD in the MMR system, and there may be no homolog of these two proteins in the eukaryotic genome. Therefore, some divergence in the MMR system from strand discrimination and the nicking process might occur between prokaryotes and eukaryotes.

A recent increase in our knowledge of the eukaryotic MMR system provides some understanding of this divergence. In mammalian cell extracts, mismatches provoke initiation of excision at pre-existing nicks in exogenous DNA substrates with high efficiency and specificity. The molecular nature of eukaryotic MMR could be assessed using cell extract assays in vitro, and components of the eukaryotic MMR system have been identified with depletion and complementation assays using cell extracts. One protein, identified in this way, is proliferating cell nuclear antigen (PCNA). PCNA is known to function as a processivity factor for replicative polymerase, but some mutations in the PCNA gene result in mutator phenotypes, and its interactions with MSH2 and MLH1, and with MSH6, suggest that it functions in MMR. PCNA has biochemical activity that increases the binding of MutSa to mismatched DNA; the interactions between PCNA and MSH6 are essential for this biochemical activity, which suggests that PCNA might play a role in MMR at the mispair recognition stage. PCNAhas been proposed to function in the mismatch recognition stage of MMR by helping MutSa search for mismatched DNA or increasing the mismatchbinding specificity of MutSa. One intriguing point about the role of PCNA in eukaryotic MMR is that the requirement for PCNA depends on the direction of the nick in the in vitro MMR assay. Although PCNA is required for mismatch-provoked excision directed by a 3’ strand break in HeLa nuclear extracts, it is not essential for excision directed by a 5’ nick. Moreover, whereas 3’ nick-directed excision iscompletely abolished by the inhibition of PCNA, 5’ nick-directed excision is affected only minimally.

Finally, a mismatch-provoked 5’ to 3’ excision reactioncan be reconstituted in a purified system that comprises only MutSa, MutLa, ExoI and replication protein A (RPA), without PCNA, and the process is similar to that observed in nuclear extracts. RPA, the eukaryotic single-stranded DNA-binding protein, has been shown to enhance excision and stabilize excision intermediates in crude fractions. The activities of ExoI are described below.

Genetic studies in yeast, and biochemical studies of MMR activity in cell extracts, indicate that eukaryotes use a mechanism similar to prokaryotes, with both 3’ to 5’ and 5’ to 3’ exonuclease activities for mismatch correction. ExoI, a 5’ to 3’ exonuclease, was found to play a role in mutation avoidance and mismatch repair in yeast, and its physical interaction with MSH2 and MLH1 also support a role in MMR. Intriguingly, the mammalian ExoI was reported to be involved in both 5’- and 3’ nick-directed excision in extracts of mammalian cells, but how ExoI can have a 3’ to 5’ exonuclease activity was unclear. Recent research by the Modrich group provides a plausible answer to this question [49]. They reconstituted mismatch-provoked excision, directed by a strand break located either 3’ or 5’ to the mispair, in a defined human system using purified human proteins. In the presence of the eukaryotic clamp loader replication factor C (RFC) and PCNA, 3’ to 5’ excision was supported by MutSa, MutLa, ExoI and RPA. Moreover, RFC and PCNA act to suppress 5’ to 5’ excision when the strand break that directs hydrolysis is located 3’ to the mismatch, which suggests that the polarity of mismatch-provoked excision by ExoI is regulated by PCNA and RFC. Once the strand is excised beyond the mismatch, DNA resynthesis occurs by the activity of polymerase d in the presence of PCNA and RPA. The remaining nick is then sealed by an as-yet-unidentified ligase, completing the repair process.

Mutation

Mutation is a permanent change in the DNA sequence that makes up a gene. Mutation range in size from one DNA base to a whole chromosome change.

Gene mutation can occur in two ways:

  • Germ line Mutation or hereditary Mutation – Mutations that can be passed down (are hereditary) and are present in all cells of an individual, including the germ cells.
  • Acquired or sporadic Mutations – Mutations caused by genetic changes that happen mostly by chance and are not inherited. They are acquired sometime during a person’s lifetime. These genetic changes are called sporadic (spontaneous) or acquired mutations. Most of these mutations occur in somatic cells. Somatic cells are any cells in the body other than egg and sperm cells (germ cells). Mutations in somatic cells cannot be passed down from a parent to a child.

These changes can be caused by environment factors such as ultraviolet radiation from the sun, or can occur due to the errors that occur during cell division. Errors in replication and damage to DNA have two consequences:

  • Permanent change to DNA, which can alter the coding sequence of a gene or its regulatory sequences.
  • Some chemical alterations to the DNA prevents its use as a template for replication and transcription.

The perpetuation of the genetic material from generation to generation depends on the maintaining rates of mutation at low levels. High rates of mutation in the germ line would destroy the species (lead to species extinction), and high rates of mutation in the soma would destroy the individual (leads to development of new species along with the existing one).

Sources of Mutation:

  1. Inaccuracy in DNA replication: The enzymatic machinery for replicating DNA attempts to cope with the mismatch incorporation of incorrect nucleotides through a proofreading mechanism, but some errors escape detection.
  2. Chemical Damage: Natural and unnatural chemicals and radiations break its backbone and chemically alters its bases. Example: UV radiation, X- ray and Gamma Radiation.
  3. Transposons: It is a DNA sequence that can change its position within the genome, sometimes creating or reversing mutations and altering the cell’s genome size. It can insert large portion of base pairs (may be even thousand bp) from one place to another.

Types of Mutation:

Mutation may  be gross or large ( which contain large portion or whole chromosome), or point (mutations not visible as cytological abnormalities), which can involve change in just a single nucleotide pair in DNA.

A. Base Pair (Nucleotide Pair) Substitutions:

Base pair substitution are the simplest mutation and involve switching of one base pair for another. These are of two types:

  • Transitions (Purine to purine or pyrimidine to pyrimidine substitution)
  • Transversions (Purine to pyrimidine or pyrimidine to purine substitution)

The consequences of base substitution mutations in protein coding regions of a gene depends on the substitution and its location. They may be

Silent: not resulting in a new amino acid in the protein sequence, eg GCA or GCG codons in mRNA both mean Arginine.

Missense Mutation: A base substitutaion which result in amino acid substitution. Eg. CTC in the DNA sense strand will specify glumate residue in the protein; this is altered to CAC in the DNA or GUG in the mRNA, resulting in a Valine residue in the beta-globulin protein chain causing sickle-cell anemia.

Nonsense Mutation: Base substitutions in a protein coding region may mutate an amino acid codon to termination code or vise versa, which results in a prematurely shortened protein.

Base substitution mutation may also occur in promoters or 5′ regulatory regions of genes or in introns and may affect their transcription, translation, or splicing. Many of the beta-thalassemias are the result of these types of non-structural mutations that may affect the level of expression of the globin genes. All of the types of the mutation described above have been observed in human globin genes. Their consequences depends on what they do to the level of expression of the gene products and/or on what amino acid substitution may have occured and where it is in the protein.

B. Frameshift Mutation (Insertions, Deletions and Duplications)

These result from insertion or deletion of one or more nucleotides in the coding region of the gene. This causes an alteration of the reading frame: since codons are groups of three nucleotides, there are three possible reading frames for each gene although only one is used. A mutation of this sort changes all the amino acids downstream and is very likely to create a nonfunctional product since it may differ greatly from the normal protein.

C. Repeat Expansions

Nucleotide repeats are short DNA sequences that are repeated a number of times in a row. For example, a trinucleotide repeat is made up of 3-bp sequences. A repeat expansion is a mutation that increase the number of times that the short DNA sequence is repeated. This type of mutation can cause resulting protein to function improperly.

D. Changes in Chromosomes

Changes that affect entire chromosomes or segments of the chromosomes can cause problems with growth, development, and function of the body’s systems. These changes can affect many gene along the chromosome and alter the proteins made by these genes. Conditions caused by the change in the number or structure of chromosomes are known as chromosomal disorders. these changes can occur during the formation of productive cells or in early fetal development. Many cancer cells also have changes in their chromosome number or structure. These changes most often occur in somatic cells during a person’s lifetime.

Proneness to Mutations

DNA sequences contain regions which are more prone to mutation. These mutation-prone sequences are repeats of simple di, tri, or teranucleotide sequences, which are known as DNA microsatellites.

 

Replication in Prokaryotes

The synthesis or replication of DNA molecule can be divided into three stages

  1. Initiation (Formation of Replisome)
  2. Elongation (Initiation of synthesis and elongation)
  3. Termination

I) Initiation

The replication begins at a specific initiation point called OriC point or replicon. (Replicon: It is a unit of the genome in which DNA is replicated; it contains an origin for initiation of replication)  It is the point of DNA open up and form open complex leading to the formation of prepriming complex to initiate replication process.

The OriC site is situated at 74″ minute near the ilv gene.  The OriC site consists of 245 basepairs, of which three of 13 basepair sequence are highly conserved in many bacteria and forms the consensus sequences (GATCTNTTNTTTT).  Close to OriC site, there are four of 9 basepair sequences each (TTATCCACA).

The sequence of reactions in the initiation process is as follows:

a) Dna A protein recognizes and binds up to four 9bp repeats in OriC to form a complex of negatively supercoiled OriC DNA wrapped around a central core of Dna A protein monomers.  This process requires the presence of the histone like HU or 1 HC proteins to facility DNA bending.

 

b) Dna A protein subunits then successively melt three tandemly repeated 13bp segments in the presence of ATP at >=22*C (open complex).

c) The Dna A protein then guides a Dna B – Dna C complex into the melted region to form a so called prepriming complex.  The Dna C is subsequently released.  Dna B further unwinds open complex to form prepriming complex.

d) DNA gyrase, single stranded binding protein (SSB), Rep protein and Helicase – II are bound to prepriming complex and now complex is called as priming complex.

e) In the presence of gyrase and SSB, helicases further unwinds the DNA in both directions so as to permit entry of primase and RNA polymerase.  Then RNA polymerase forms primer for leading strand synthesis while primase in the form of primosome synthesis primer for lagging strand synthesis.

f) To the above complex, DNA polymerase – III will bind and forms replisome.

REPLISOME:  It is the multiprotein structure that assembles at the bacterial replicating fork to undertake synthesis of DNA.  It contains DNA polymerase and other enzymes.

II) ELONGATION:

Now the stage is set for the initiation of synthesis and the elongation to proceed.  But this occurs in two mechanistically different pathways in the 5′–>3′ template strand and 3′–>5′ template strand.

a) Initiation of synthesis and Elongation on the 5′–>3′ template (synthesis of leading strand) (If replication fork moves in 3′–>5′ direction)

The DNA daughter strand that is synthesized continuously on 5′–>3′ template is called leading strand.  DNA pol-III synthesizes DNA by adding 5′-P of deoxynucleotide to 3′-OH group of the already presenting fragment.  Thus chain grows in 5′–>3′ direction.  The reaction catalyzed by DNA pol-III is very fast.  The enzyme is much more active than DNA pol – I and can add 9000 nucleotides per minute at 37*C.  The RNA primer that was initially added by RNA polymerase is degraded by RNase.

b) Initiation of synthesis and Elongation on 3′–>5′ template when fork moves in 3′–>5′ direction (Synthesis of lagging strand) 

The daughter DNA strand which is synthesized in discontinuous complex fashion on the 3′–>5′ template is called lagging strand.  It occurs in the following steps:

i) Synthesis of Okazaki fragment:

To the RNA primer synthesized by primosome, 1000-2000 nucleotides are added by DNA pol-III to synthesis Okazaki fragments.

ii) Excision of RNA primer:

        When the Okazaki fragment synthesis was completed up to RNA primer, then RNA primer was removed by DNA pol – I using its 5′–>3′ exonuclease activity.

iii) Filling the gap (Nick translation)

        The gap created by the removal of primer, is filled up by DNA pol – I using the 3′-OH of nearby Okazaki fragment by its polymerizing activity.

iv) Joining of Okazaki fragment: (Nick sealing)

        Finally, the nick existing between the fragments are sealed by DNA ligase which catalyze the formation of phosphodiester bond between a 3′-OH at the end of one strand and a 5′ – phosphate at the other end of another fragment.  The enzyme requires NAD for during this reaction.

III) TERMINATION:

 

Termination occurs when the two replicating forks meet each other on the opposite side of circular E.Coli DNA.  Termination sites like A, B, C, D, E and F are found to present in DNA. Of these sites, Ter A terminates the counter clockwise moving fork while ter C terminates the clockwise moving forks.  The other sites are backup sites.   Termination at these sites are possible because, at these sites tus protein (Termination utilizing substance) will bound to Dna B protein and inhibits its helicase activity.  And Dna B protein released and termination result.

After the complete synthesis, two duplex DNA are found to be catenated (knotted).  This catenation removed by the action of topoisomerase.  Finally, from single parental duplex DNA, two progeny duplex DNA synthesized.

REGULATION OF PROKARYOTIC REPLICATION:

Especially initiation of replication is regulated.  Dna A protein when available in high concentration then ratio of DNA to cell mass is quiet high but at low Dna A concentration, the ratio found to be low.  This shows that Dna A protein regulates the initiation of replication.

The sequence most commonly methylated in E.Coli is GATC including in three of 13mer sequence.  Thus, the observation that E.Coli defective in the GATC methylation enzyme are very inefficiently replicated, suggests that the DNA replication trigger also responds to the level of OriC methylation.

Replication in Eukaryotes

Eukaryotic replication occurs during s-phase of cell cycle.  Replication usually occurs only one time in a cell.  Replication in eukaryotes occur in five stages namely,

  1. Pre-initiation
  2. Initiation
  3. Elongation
  4. Termination
  5. Telomerase function
  1. Pre-initiation:

Actually during pre-initiation stage, replicator selection occurs.  Replicator selection is the process of identifying the sequences that will direct the initiation of replication and occur in G1 phase. and occurs in Gl (prior to S phase). This process leads to the assembly of a multiprotein complex at each replicator in the genome. Origin activation only occurs after cells enter S phase and triggers the Replicator – associated protein complex to initiate DNA unwinding and DNA polymerase recruitment.  Replicator selection is mediated by the formation of pre-replicative complexes (pre-RCs). The first step in the formation of the pre-RC is the recognition of the replicator by the eukaryotic initiator, ORC (Origin recognition Complex). Once ORC is bound, it recruits two helicase loading proteins (Cdc6 and Cdtl). Together, ORC and the loading proteins recruit a protein that is thought to be the eukaryotic replication fork helicase (the Mem 2-7 complex). Formation of the pre-RC does not lead to the immediate unwinding of origin DNA or the recruitment of DNA polymerases. Instead the pre-RCs that are formed during Gl are only activated to initiate replication after cells pass from the Gl to the S phase of the cell cycle.

rep3.14

 

  1. Initiation:

Pre-RCs are activated to initiate replication by two protein  kinases namely Cdk (Cyclin Dependant Kinase) and Ddk (Ddt4 Dependant Kinase). Kinases are proteins that  covalently attach phosphate groups to target proteins. Each of these kinases is inactive in Gl and is activated only when cells enter S phase. Once activated, these kinases target the pre-RC and other replication proteins. Phosphorylation of these pro-proteins results in the assembly of additional replication proteins at the origin and the initiation of replication.

 

These new proteins include the three eukaryotic DNA polymerases and a number of other proteins required for their recruitment. Interestingly, the polymerases assemble at the origin in a particular order. DNA Pol d and e associate first, followed by DNA Pol a/primase. This order ensures that all three DNA polymerases are present at the origin prior to the synthesis of the first RNA primer (by DNA Pol a/primase).   Once present at the origin, DNA Pol a/primase synthesizes an RNA primer and briefly extends it. Thus initiation of replication started.

rep3.15

 

  1. Elongation:

 

The resulting primer-template junction is recognized by the eukaryotic sliding clamp loader (RF-C), which assembles a sliding clamp (PCNA) at these sites. Either DNA Pol d or e recognizes this primer and begins leading strand synthesis. After a period of DNA unwinding, DNA Pol a/primase synthesizes additional primers, which allow the initiation of lagging strand DNA synthesis by either DNA Pol d or e. In the diagram, Pol d was used for leading strand and Pol e was used for lagging strand synthesis.  DNA Pol e possess activity to remove primer and fills the gap with DNA like DNA Pol I in prokaryotes.  SSB like activity was played by replication protein A (RP A) which is denoted as accessory factors during replication.

 

  1. Termination:

 

When the replication forks meet each other, then termination occurs.  It will result in the formation of two duplex DNA.  Eventhough replication terminated, 5’ end of telomeric part of the newly synthesized DNA found to have shorter DNA strand than the template parent strand.  This shortage corrected by the action of telomerase enzyme and then only the actual replication completed.

  1. Terlomerase Function:

In Linear eukaryotic chromosome, once the first primer on each strand is remove, then it appears that there is no way to fill in the gaps, since DNA cannot be extended in the 3′–>5′ direction and there is no 3′ end upstream available as there would be in a circle DNA.  If this were actually the situation, the DNA strand would get shorter every time they replicated and genes would be lost forever.

Elizabeth Blackburn and her colleagues have provided the answer to fill up the gaps with the help of enzyme telomerase.  So, that the genes at the ends, are conserved.  Telomerase is a ribonucleoprotein (RNP) i.e. it has RNA with repetitive sequence.  Repetitive sequence varies depending upon the species example Tetrahymena thermophilia RNA has AACCCC sequence and in Oxytrica it has AAAACCCC.  Telomerase otherwise known as modified Reverse Transcriptase.  In human, the RNA template contains AAUCCC repeats.  This enzyme was also known as telomere terminal transferase.

The 3′-end of the lagging strand template basepairs with a unique region of the telomerase associated RNA.  Hybridization facilitated by the match between the sequence at the 3′-end of telomere and the sequence at the 3′-end of the RNA.  The telomerase catalytic site then adds deoxy ribonucleotides using RNA molecule as a template, this reverse transcription proceeds to position 35 of the RNA template.  Telomerase then translocates to the new 3′-end by pairing with RNA template and it continues reverse transcription.  When the G-rich strand sufficiently long, Primase can make an RNA primer, complementary to the 3′-end of the telomere’s G-rich strand.   DNA polymerase uses the newly made primer to prime synthesis of DNA to fill in the remaining gap on the progeny DNA.  The primer is removed and the nick between fragments sealed by DNA ligase.

rep3.17

DNA replication in Prokaryotes

All living cells are capable of giving rise to a new generation of cells by undergoing

DNA replication and cell division. During cell division in eukaryotic cells, the replicated DNA is equally distributed between two daughter cells.

During replication process the hydrogen bonds between complimentary strands break and allow the DNA helix to unzip. Each strand of unwound DNA acts as template to build complimentary strand.

The process of DNA replication:

1. The first step in the process of DNA replication is to unwind the DNA. An enzyme known as helicase helps in breaking the hydrogen bonds and unwinding the DNA.

2. Even though the strands are separated they have a tendency of annealing back again due to complimentary nature of base pairs. Single stranded binding proteins (SSBs) bind to the exposed DNA and keep them apart by blocking the hydrogen bonding.

DNA replication

3. Another enzyme gyrase helps to release the tension in the separated strands by cutting and resealing them.

4. DNA is unwound at multiple locations forming bubbles known as replication bubbles. The junction where DNA is still attached is known as replication fork.

Replication bubble

5. DNA polymerase III enzyme helps in synthesizing the complimentary strand using template strand as a guide.

6. DNA polymerase III can function only under certain conditions

i) it synthesizes DNA in 5’ to 3’ direction

ii) it require the presence of RNA primer to initiate complimentary strand

7. RNA primer is synthesized by primase enzyme.

8. Complimentary stand can be made continuously on one of the template strand known as leading strand. Other strand is known as lagging stand.

replication showing okazaki fragments

9. On leading strand one RNA primer is attached at 3’ end and complimentary stand is made uninterrupted.

10. On lagging stand multiple RNA primers are attached to template strand and synthesize small fragments of complimentary strands. These fragments are known as Okazaki fragments.

11. RNA primers are removed by DNA polymerase I and are replaced with appropriate deoxyribonucleotides.

12. Ligase enzyme joins all Okazaki fragments together by forming bonds between them.