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).
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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.