Cisplatin

Cisplatin, also known as cisplatinum or cis-diamminedichloroplatinum(II), is a chemotherapy drug used to treat various types of cancer including sarcomas, lymphomas, germ cell tumors and some carcinomas (e.g. small cell lung cancer, ovarian cancer).

This molecule was first created in mid-19th century, being its discoverer Michel Peyronie.

Cisplatin belongs to a class of platinum-containing anti-cancer drugs and it was the first medicine developed in this class.

The molecule’s platinum complex reacts in vivo, binding with DNA forming intra-strand crosslinks which causes a conformational change in DNA affecting its replication. Cisplatin cytotoxicity is increased during S-phase and causes cell cycle to cease in the G2-phase, ultimately triggering apoptosis, i.e. programmed cell death.

Other mechanisms of cisplatin’s cytotoxicity include decreased ATPase activity, mitochondrial damage and altered cellular transport mechanisms.

Cisplatin-induced cellular destruction can result in tumor lysis syndrome (TLS) which includes hyperkalemia, hyperphosphatemia, hyperuricemia, hypocalcemia, which means, respectively, high concentrations of K⁺, phosphate and uric acid in the blood, and low concentration of serum calcium, also in the blood.

Replication by strand displacement

·        Replication can occur in various numbers of ways. One of which is strand displacement replication. This is a type of replication consists in the replication of just one ssDNA molecule from a DNA template.

·         DNA replication is a semi-conservative replication, due to the fact that two double stranded DNA (dsDNA) strand are formed from one dsDNA strand and these two “daughter” dsDNA possess one of the strands from the “mother” DSdna. In other words, in a cell possessing one mother dsDNA, the DNA strand present in the cell is 100% of the mother dsDNA, but after replication, of all the DNA strand in the cell, 50% of it will be from the mother dsDNA and after another replication the percentage of mother dsDNA strands is 25% and so on.

  

·         Strand displacement replication occurs do to the fact that DNA polymerase III not only catalyzes replication from ssDNA templates, but catalyzes as well a replication process in which a flapped strand of DNA is combined with a complementary DNA strand. In this replication type, there are three strands of DNA: strand 1, strand 2 and strand 3. Strands 2 and 3 are connected to each other and strand 1 is complementary to strand 3. Both strand 1 and 3 possess small complementary nucleotidic sequences that strand 2 does not. What happens is that these small nucleotidic sequences of strands 1 and 3 can combine. Since these sequences are small, their connection is easily achieved, but can be torn apart just as easily. When this connection lasts long enough, the first non-connected nucleotide of strand 1 can link itself to its complementary nucleotid in strand 3. This process removes the nucleotide of strand 2 that was linked in that spot. This process repeats itself until strand 1 displaces strand 2 from strand 3.  

Topotecan

Topotecan is a chemotherapeutic product that belongs to the antineoplastic agents, substances that inhibit or prevent the decontrolled proliferation of cells. It is a semi-synthetic medicine that is synthetized through camptothecin and has activity in a wide range of tumor types.

Topotecan is used on cancer therapy because it inhibits the topoisomerase I, which controls DNA topology by relieving the strain in DNA supercoils during replication, and causes single-strand DNA breaks which inhibit DNA functionality. This process prevents mitosis or leads to cell death by generating double-stranded DNA breaks when DNA replication occurs. Cancer cells divide faster than normal cells, so they are more susceptible to be affected by topotecan.

This medicine´s activity is greatest when administrated continuously over a long period of time, and it has been successfully combined with other antineoplastic agents, such as cisplatin.

Telomerase

Telomerase is an enzyme that preserves the ends of eukaryotic chromosomes, preventing their shortening, through the synthesis of telomeric repeat sequences. This is a ribonucleoprotein since it is composed of an RNA subunit (hTR or hTERC) and a protein subunit (hTERT) – catalytic part (reverse transcriptase acting on the 3 'end of the DNA chain). It has its own mold (template) of DNA. The RNA portion of human telomerase has approximately 450 nucleotides and the mold region, containing eleven, is complementary to the telomeric sequence 5’-TTAGGG-3’. In all organisms, the produced sequence is rich in guanine.
That enzyme is active only in stem cells, breeding and embryonic, it is also found in cancerous cells. In the latter case, the telomerase activation occurs unusual and uncontrollably. Since prevents shortening of telomeres allows cells to divide an infinite number of times without loss of genetic information, conferring immortality to them.
The telomerase enzyme is considerate a biologic clock, a marker indicating that cellular senescence will inevitably be installed, causing aging in cells that exhibit telomerase activity.

DNA Polymerase Gamma (γ)

The DNA polymerase γ (Pol- γ) is an enzyme responsible for the replication and repair of the mitochondrial genome in eukaryote cells, and despite being a mitochondrial enzyme it is coded by nuclear genes.

This enzyme, in humans for example, is a 195 kDa heterotrimer, made up of one catalytic subunit (coded by the gene POLG, situated in chromosome 15q25 – long arm of chromosome 15, region 2, band 2) and an dimeric accessory subunit (coded by the gene POLG2 situated in the chromosome 17q- long arm of chromosome 17). The catalytic subunit possesses only one exonuclease activity that is the 5’-3’ exonuclease activity (proofreading) and a 5’dRP lyase activity, that is the feature responsible for the repair of nucleotide base pairs, by the base excision repair mechanism. The accessory subunit  acts as a DNA binding factor that confers high processivity by increasing the enzyme’s affinity for template DNA.  DNA polymerase γ has a high base-substitution fidelity and is relatively precise for short repeat sequences but longer homopolymer segments tend to yield replication slippage by DNA polymerase γ.

Unlike nuclear DNA, that only replicates during cell division, mitochondrial DNA is continually being recycled, independently of the cell cycle, and as such, mutations in the genes responsible for the coding of the DNA Polymerase γ will have drastic consequences in the individuals where those mutations occur.  For example, mutations that lead to a loss of the DNA polymerase γ exonuclease activity (mutation on the POLG gene) will lead to a much more accelerated aging, this because the mitochondrial genome codes for 13 polypeptide subunits of the respiratory chain. If the respiratory chain is compromised, the flow of electrons will yeld free radicals that will contribute to a greater cell oxidation, and beyond that the ATP formation will be also compromised. This phenomenon’s are responsible, for example, the Alper’s disease.

   

The DNA polymerase γ activity is still poorly understood, however it is known that the Nuclear Respiratory factor-1 (NRF-1) is a transcription factor that regulates the expression of many mitochondrial proteins, by binding to promoter regions of POLG, POLG2 and mtTFA (mitochondrial transcription factor). The binding of NRF-1 is related to the ATP levels.

Cell cycle checkpoints

Cell cycle checkpoints are control mechanisms that regulate cell division and prevent cells from continuing through the cycle if the events of the preceding phase have not been completed. The main control points in mammals are:

•       At the end of G1 phase (restriction point) the cell checks the quality of the DNA, the presence of specific growth factors (p.e. fibroblast growth factor) and cell size. Arrest at that checkpoint allows repair of damaged DNA before the cell enters the replication phase. If DNA can’t be repair the cell induces apoptosis. The phase of arrest in which the cell doesn’t continue its cycle is G₀. This checkpoint is controlled by Cyclin D-CDK 4/6 and Cyclin E-CDK 2;
•          At the S phase, continual control of the integrity of DNA allows that mutated bases are not replicated and the repair of possible errors that occur during replication. The proteins involved in this damage detection are DNA pol ε, PCNA and RFC. This checkpoint is controlled by Cyclin A-CDK 2;
•          At the end of G2 phase , cells check if there are conditions for mitosis (cell size, quality of DNA and existence of nutrients necessary for the mitotic phase) and prevent the initiation of mitosis before DNA replication is completed and until there are conditions for cell division (cell size and existence of necessary nutrients). If there are no conditions, cells enter in a quiescent state where DNA repair or apoptosis can occur. This checkpoint is controlled by Cyclin A-CDK1;
•     At the beginning of anaphase, spindle assembly checkpoint stops mitosis if the chromosomes are not well aligned and its centromeres are not properly attached to the microtubules and thus, not prepared for equal distribution. This checkpoint is controlled by the anaphase-promoting complex/cyclosome (APC/C).
• 
  

Fig.1 The cell cycle checkpoints.

In case of a failure in these checkpoints, cells with damaged DNA can proliferate at higher rates than normal cells, becoming neoplasic.

For this reason the studies of these checkpoints in the cell cycle are important for the production of new drugs in the treatment of problems like  cancer.

GEOFFREY M. COOPER, ROBERT E. HAUSMAN , (2007). THE CELL: A Molecular Approach . 4th ed. e.g. England. Sunderland (MA): Sinauer Associates.

STEM CELLS

Stem cells are undifferentiated cells able to divide and differentiate into any kind of specialized cells. When this division occurs it originates two daughter cells, one that remains as stem cell to assure their maintenance and a specialized one such as a muscle cell or a red blood cell.

Usually, stem cells come from two main sources: embryos formed during the blastocyst stage within the embryonic development also called embryonic stem cells or from adult tissue that is called adult stem cells.

When compared with others cells types the stem cells are distinguished by two important and singular characteristics. In first place these cells are able to renew themselves through cell division even after prolonged period of inactivity. Another characteristic is that under certain experimental or physiologic conditions they can be induced to become a specific organ or tissue with specialized cells.

Ribonucleotide reductase

 

          Ribonucleotide reductases (RNRs) is an enzyme that catalyze the conversion of ribonucleotides to deoxyribonucleotides in all organisms and play an essential role in DNA replication and repair.
          The substrates for RNR are UDP, GDP, ADP and CDP. Because of their central role, they are also successful targets of several drugs used clinically in the treatment of number of malignancies. This happens because Ribonucleotide reductase inhibitors are a family of anti-cancer drugs that interfere with the growth of tumor cells by blocking of doxyribonucleotides (building blocks of DNA) .

 

 

The different functions of different types of RNR and the the action local.





Ribonucleotide reductase action on DNA synthetis

Primossome

Primosome is a protein complex that creates RNA primers during DNA replication. This complex is formed by seven proteins: DnaG primase, DnaB helicase, DnaC helicase, DnaT, Pri A, Pri B and Pri C. During de DNA replication, the primossome is used, in the replication fork, once in the leading stand and multiplied times in the lagging strand, one for each Okazaki fragment.

 Pre-priming complex (E. coli)

The pre-priming complex is formed, at the oriC, in the initiation phase of E. coli DNA replication.

The association of dnaB protein with oriC represents the first step at which binding of DNA a protein to the origin results in the assembly of replication enzymes for the initiation of DNA synthesis.

DnaB and dnaC protein form a complex, formed in solution with ATP.  The dnaA protein guides a dnaB - dnaC complex into the melted region to form a pre-priming complex.

The dnaC protein is not stably maintained in the complex, in which it inhibits the helicase activity of dnaB protein and so must be ejected for replication proceeds.

The pre-priming intermediate is stable, isolatable, and resistant to low temperature.

Rolling Circle Replication

1- Rolling circle replication's steps

In rolling circle replication the DNA is in a circular formation. Instead of both strands being a template strand, only one strand is. Nickase makes a nick in one of the strands, the outer, and this creates a 5’ phosphate and a 3’ hydroxyl. The 3’ works as a primer for the polymerase allowing it to function.  Such action will push the old “nicked” strand off of the template. When, in the long template, the sites cos are recognized a endonuclease cleaves the DNA thus ending replication.

Guanazole is a cytostatic triazole derivative. This molecule belongs to a group of highly explosive materials, and they are vulnerable to heat, impact and friction.

Guanazole can be used in many applications, such as Chemistry and Medicine, among others. It has anti-tumor activity, by inhibition of DNA synthesis. This process occurs because this molecule inhibits ribonucleotide reductase, enzyme intervenient in the deoxyribonucleotides synthesis.

                                                  
                                                Estrutura  quimica da molécula de Guanazole

Plectonemic Structure

Plectonemic is any tertiary structure in a polymeric molecule with supercoil strands in a regular and simple form, such as supercoiled DNA.

This type of supercoiling – form shown by DNA in vivo – doesn’t produce sufficient compaction to package DNA in the cell.

In the origin of plectonemic structures there are two interwound helical filaments whose geometry is characterized by a superhelical filament angle and radius. Due to this, the superhelical angle and the twisting moment in the filaments control the action of some enzymes like topoisomerases, RNA polymerase, helycases and DNA polymerases.

Another type of supercoiling is the solenoidal form which is shown in underwound DNA. Both structures are forms of negative supercoiling and are interconvertible, though they’re two different structures.

The main advantage of plectonemic structure is it’s stability in solution, although solenoid forms can be stabilized when present in high levels in tumor cells.

Fig.1 A plectonemic helix with radius r, pitch p and opening angle a.

Image source:
Job Ubbink, Theo Odjik, Electrostatic-Undulating Theory of  Plectonemically Supercoiled DNA, Biophysical Journal, Volume 76, Issue 5 (May 1999), pp. 2502-2519, Faculty of Chemical Engineering and Materials, Delft University of Technology, 2600 GA Delft, the Netherlands

Cell senescence

Cell senescence is observed when cells stop dividing. This happens when telomeres, which protect the end of the chromosome, become progressively shorter reaching the Hayflick limit (limit to cell division capacity), meaning that human cells can only divide a finite number of times. In Cell senescence includes progressive and irreversible loss of cellular functions leading to the death of some cells.

Whenever a cell division occurs, the telomere loose length and become shorter. When they reach a minimum size, chromosome duplication ceases to occur, disabling cell division.

During this process, beta-galactosidase (enzyme responsible for hydrolysis of lactose in galactose and glucose) is detected in cell’s lysosomes, indicating senescent cells.

This process is not yet fully known, raising some doubts, which leads to the emergence of several theories. Studies show that cellular senescence may be a manifestation of loss of telomerase activity. This ribonucleoprotein is an enzyme that adds DNA sequence repeats in telomere region and thereby restores the ability of cell multiplication and retards the aging of tissues. During development, the telomerase function decline and telomeres become shorter. 

Cohesin

·     Cohesines are a member of a big protein family called SMC (Structural Maintenance of Chromosomes), who have a very important role in structural and functional organization of chromosomes.

·      SMC proteins have a crucial role in chromosome segregation and DNA repair. These proteins are interesting because of, among other aspects, their unique structure. They are dimers formed by two long coiled-coil motifs (a rod like structural shape that is formed by two long α-helices twisted around each other) connected by a non-helical sequence. An ATPase domain is created by folding a SMC monomer back to itself. This folding also creates a hinge domain at the other end. In this hinge domain, two monomers associate with each other, creating a V-shaped molecule. There are many possible structures for the SMC to possess, thanks to their high flexibility of dimers conformation.

·      Cohesines are composed by four subunits: Scc1, Scc3, Smc1 and Smc3. These last two subunits are members of SMC protein family, having therefore, ATPase domains in one end each. The two ATP domains are able to bind in the presence of ATP, thus resulting a ring-shaped structure. Scc1 and Scc3 (Spirochetal Coiled-Coil) are responsible for this binding and are also responsible of maintaining and stabilizing it. Scc1 also plays and important role in chromosome replication as it controls the separation of sister-chromatids. The cohesin ring structure is important as it keeps the sister-chromatids together during metaphase, ensuring that they travel to opposite polls of the cell (both in mitosis and meiosis). The structure facilitates as well, the spindle attachment onto chromossomes (process responsible for chromosome segregation to cell-daughters during cell division) and DNA damage repair.

Fig1: Cohesin structure

The following link is a short animation that explains the role of cohesin in cell division:

http://highered.mcgraw-hill.com/sites/9834092339/student_view0/chapter10/the_function_of_cohesin.html

Apoptosis

Apoptosis is a mechanism of programmed cell death involved in the regulation of tissue maintenance and development of organisms. It is a defense mechanism to remove excess or dangerous cells.

In this process, the chromatin condenses, cells individualize and many organelles remain intact and metabolically active for a long period. The nuclear and plasma membranes are destroyed and the nucleus is then broken up into fragments that are surrounded by the cytoplasmic membrane, producing apoptotic bodies that are degraded by macrophages. This mechanism is very distinct of necrosis, because it has no inflammatory reaction.

There are extrinsic and intrinsic apoptosis. The extrinsic pathway is triggered by the ligation of extracellular apoptotic signals to receptors in plasma membrane that lead to activation of proteins caspases in a sequence called cascade that activate other caspases or the release of cytochrome c  by the mitochondria. Intracellular apoptotic signals activate the mitochondria to release cytochrome c that indirectly activate many caspases. These proteins activate endonucleases and proteases that degrade DNA and proteins, leading to morphological changes of cells, formation of apoptotic bodies and, ultimately, cell death.

Apoptosis is a reversible process, because the blocking of apoptotic genes enhance cell survival when they are subjected to weak apoptotic signals, mutations in killer genes allow the survival of programmed cells and elimination or inhibition of macrophages result in the survival of cells.

Replicatio Factor C (RFC)

The replication factor C (Replication factor C - RFC) is a complex-key indirect involved in recruitment of the polymerase to near the replication fork and recruitment and correct positioning of complex PCNA. Composed of five subunits very conserved in eukaryotes, the complex RFC presents specific affinity for the primer-template region. The binding to the DNA molecule is ATP-dependent.

 

 

 

Fig1-Eukaryotic DNA replication: Step 1: primer synthesis by DNA polymerase - alpha (Pol-alpha); step 2: replication factor C (RFC) displacement of DNA polymerase and recruitment of proliferating cell nuclear antigen (PCNA); step 3: elongation by the newly recruited DNA polymerase-delta  holoenzyme (Pol-delta); step 4: strand displacement by Pol-delta ; step 5: cutting of the 5' displaced flap by flap endonuclease 1 (Fen1); and step 6: sealing by DNA ligase I (LigI).

 

Adriana Lima
Alexandra Meira
Bruno Oliveira

 

γ-complex

For E. coli, γ-complex is a component from the enzyme DNA polymerase III, made up of five subunits and is known by interacting with the β subunit (sliding clamp).

It’s necessary for the regulation of the attachment and removal of the DNA polymerase III from the DNA template. This function is essential for the process of attachment and detachment of the enzyme during lagging strand replication, requiring ATP. This phenomenon occurs continuously from the beginning to the end of the Okazaki fragments.

DNA Polymerase III - γ-complex