DNA REPLICATION TO CONSERVE OR NOT TO CONSERVE NOVEMBER 26, 2012 CAPE UNIT 1 BIOLOGY MRS. HAUGHTON

DNA REPLICATION is all about copying a new DNA strand. This is important when cell division occurs in making new cells. The replication has to be carefully done so that the cells in an organism contain the same arrangement of DNA thus preventing mutations.

A lot of DNA research and DNA sequencing has been carried out to know these minute processes in a cell. The DNA replication process is essential for the survival of all life forms.

DNA replication steps occur during the inter-phase and are copied before cell division. Specialized cells like the muscles and nerve cells do not divide, thus, there is no DNA replication process carried out here.

DNA FACTS

The human DNA is up to 80 million base pairs long in a chromosome. Thus, the DNA is unwound at multiple places along its length and DNA replication steps are carried out simultaneously at many places. 

The human DNA is copied at about 50 base pairs per second. The multiple location of DNA replication process takes about 1 hour to complete. If this were not the case, then it would take about a month to finish replicating the entire DNA strand!

The DNA replication process is almost error free with the help of DNA polymerase and other simple DNA replication enzymes, that proofread the nucleotides being added to the strand.

If the nucleotides are not found to be complementary, then they are removed and a new nucleotide is synthesized. Thus, creating an error free DNA strand.

A billion nucleotides have less than one mistake. This means that copying 100 dictionaries with 1000 pages word to word, page to page and symbol to symbol, with only one mistake! High fidelity.

DNA is a marvellous coding chip, that contains all the information required for the function of a cell and the organisms. Nature has thought of every detail that helps in the growth of a species. This error free factory of nature, is one of the unmatched manufacturing units, that help produce organisms of the highest quality!

HOW DID WE FIND OUT WHAT DNA WAS FOR?

GRIFFITHS EXPERIMENTS Evidence from bacteria http://www.youtube.com/watch?v=vQOdDGM5vSg

HERSHEY AND CHASE Evidence from viruses http://www.youtube.com/watch?v=YG3d77SRWZI

When we left off we were trying to decide if DNA replicated in a conservative, semi-conservative or dispersive manner. Meselson and Stahl’s experiments with heavy (15N) and light (14N) nitrogen was the key to unlocking this. http://www.youtube.com/watch?v=ZTMyNmZrcwA

So DNA replication is semi-conservative!

IMPORTANT POINTS This is the most common method of DNA replication. It takes place in the nucleus where the DNA is present in the chromosomes.

Replication takes place in the S-phase (synthesis phase) of the interphase nucleus. The deoxyribose nucleotides needed for the formation of  the new DNA strands are present in the nucleoplasm.

DNA REPLICATION IN EUKARYOTES

Watson and Crick proposed that the two strands were capable of unwinding and separating, and acting as templates to which a complementary set of nucleotides would attach by base pairing. In this way each original DNA molecule would give rise to 2 copies with identical structures.

Kornberg showed that DNA could be synthesized in a test-tube by using a single strand of DNA as a template. He used an enzyme called DNA polymerase to do this.

Experiments showed that the nucleotides used naturally in cells have two extra phosphate groups attached. This activates the nucleotides. As each nucleotide links up to the growing DNA chain, the two extra phosphate groups break off releasing energy.

This enables the remaining phosphate group of the nucleotide to form a bond with the sugar molecule of the neighboring nucleotide

The steps are: Initiation Elongation Termination

1 Initiation occurs when the hydrogen bonds between bases of the two antiparallel strands breaks. The unwinding of the two strands is the starting point.

The splitting happens in places of the chains which are rich in A-T. That is because there are only two bonds between Adenine and Thymine (there are three hydrogen bonds between Cytosine and Guanine).

The enzyme Helicase splits the two strands. The initiation point where the splitting starts is called "origin of replication". The structure that is created is known as the "Replication Fork".

On the replication fork, the DNA replication process occurs simultaneously on each fork. The proteins that are involved in the DNA replication process are collected in one location of the cell.

This shows that the proteins do not move along the length of DNA, but the DNA is fed through the protein factory or area just like a film is fed into a projector.

The single-stranded binding proteins (SSBs) cover the DNA strands thus preventing them from annealing into a double strand. These SSBs are easily moved by the DNA polymerase enzyme.

2 The next step is the binding of RNA Primase in the the initiation point of the 3'-5' parent chain. 

RNA Primase can attract RNA nucleotides which bind to the DNA nucleotides of the 3'-5' strand due to the hydrogen bonds between the bases. RNA nucleotides are the primers (starters) for the binding of DNA nucleotides. 

3) The elongation process is different for the 5'-3' and 3'-5' template. a)5'-3' Template: The 3'-5' proceeding daughter strand -that uses a 5'-3' template- is called leading strand because DNA Polymerase I can "read" the template and continuously adds nucleotides (complementary to the nucleotides of the template, for example Adenine opposite to Thymine etc). 

b)3'-5'Template: The 3'-5' template cannot be "read" by DNA Polymerase I. The replication of this template is complicated and the new strand is called lagging strand. In the lagging strand the RNA Primase adds more RNA Primers. 

DNA polymerase III reads the template and lengthens from the primers DNA polymerase III reads the template and lengthens from the primers. The gap between two RNA primers is called "Okazaki Fragments".  The RNA Primers are necessary for DNA Polymerase I to bind Nucleotides to the 3' end of them. The daughter strand is elongated with the binding of more DNA nucleotides. 

4 In the lagging strand the DNA Polymerase I -exonuclease reads the fragments and removes the RNA Primers. The gaps are closed with the action of DNA Polymerase which adds complementary nucleotides to the gaps, and DNA Ligase which adds phosphate in the remaining gaps of the phosphate - sugar backbone. 

Each new double helix is consisted of one old and one new chain. This is what we call semi-conservative replication

5 The last step of DNA Replication is the Termination. This process happens when the DNA Polymerase reaches to an end of the strands. We can easily understand that in the last section of the lagging strand, when the RNA primer is removed, it is not possible for the DNA Polymerase to seal the gap (because there is no primer).

So, the end of the parental strand where the last primer binds isn't replicated. These ends of linear (chromosomal) DNA consists of non-coding DNA that contains repeat sequences and are called telomeres. As a result, a part of the telomere is removed in every cycle of DNA Replication. 

6 The DNA Replication is not completed before a mechanism of repair fixes possible errors caused during the replication. Enzymes like nucleases remove the wrong nucleotides and the DNA Polymerase fills the gaps.

SUMMARY

DNA REPLICATION IN PROKARYOTES

The DNA of the prokaryotic chromosome replicates as a circular structure. It is a semi-conservative as in the linear DNA of the eukaryotes. Replication begins a fixed point called the origin.

In most prokaryotes, the DNA has only one ‘origin’ point. The uncoiling of the two strands of the circular molecule begins at the point of origin and progresses in opposite directions (bidirectional). This is helped by the enzyme called DNA gyrase.

Simultaneously with the uncoiling of the original strands, a new complementary strand is being constructed on each strand. As a result, the circular DNA appears as the Greek letter q (theta) during replication. Hence this mode of replication is called q replication.

Each newly forming strand also goes on helically coiling around its circular template strand (original strand). As a consequence, by the time the replication process is over, two complete double-stranded circular DNA molecules are formed. These are exact copies of the original molecule. Also each daughter molecule has one original (parental) and the other newly formed circular strands.

RNA

ROLE OF RNA RNA exists as a single-stranded molecule in living cells. There exist 3 types. They are al involved in the synthesis of protein molecules.

They are: Messenger RNA (mRNA) Ribosomal RNA (rRNA) Transfer RNA (tRNA)

All three types are synthesized directly on DNA (template). The amount of RNA in each cell is directly related to the amount of protein synthesis.

mRNA 3-5% of RNA in each cell. Single-stranded molecule formed on a single strand of DNA during the process of transcription. Since the DNA cannot leave the nucleus to help make proteins, the RNA makes a copy of the segment of DNA required and this is what enters to cytoplasm and goes to a ribosome.

Base sequence of mRNA is a complementary copy if the DNA template strand. It varies in length depending on how much of the DNA template is copied to make a particular protein. Exists for only a short time in cell.

mRNA

rRNA 80% of RNA in each cell. Synthesized by genes present on a region of the DNA called the nucleolar organizer. After synthesis in the nucleus, it is found in the cytoplasm where it is associated with protein molecules which together form the ribosomes where the process of translation occurs.

tRNA 15% of total RNA in each cell. Acts as an intermediate molecule between the triplet code of mRNA and the amino acids sequence of the polypeptide chain.

More than 20 different tRNA molecules in a given cell carrying specific amino acids (60 have been identified). Each amino acid has its own family of tRNA molecules that transport them around.

Guanine is always at the end of the 5’-end of tRNA. The base sequence CCA (cytosine, cytosine, adenine) is always at the 3’ end. The triplet base sequence at the anticodon is directly related to the amino acid carried by the tRNA molecule at the 3’-end.

tRNA

Each amino acid is attached to its specific tRNA by its own form of the enzyme aminoacyl-tRNA synthase. This produces an amino acid-tRNA complex known as aminoacyl-tRNA.