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.

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.

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  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.

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  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.

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