Exam 2: DNA replication
DNA is a double stranded nucleic acid that forms a genetic code which contains instructions for the synthesis of new proteins. The DNA therefore directly influences the construction and behaviour of cells and the way in which they grow together to form a complete living organism. DNA also has the ability to replicate and make perfect copies, over and over again. This is called semi-conservative DNA replication and is important as the DNA inside a cell needs to double exactly such that equal amounts can be passed into the two daughter cells during cell division.
DNA has two major functions in the cell:
• Replication, in dividing cells to from exact copies
• Carrying information (genetic code) for protein synthesis in all cells
Chromosomes must make copies of themselves so that when cells divide, each daughter cell must receive an exact copy of the genetic information. This copying of DNA is called DNA replication and takes place in a cell during interphase.
DNA replication occurs as follows:
DNA is a double stranded nucleic acid that forms a genetic code which contains instructions for the synthesis of new proteins. The DNA therefore directly influences the construction and behaviour of cells and the way in which they grow together to form a complete living organism. DNA also has the ability to replicate and make perfect copies, over and over again. This is called semi-conservative DNA replication and is important as the DNA inside a cell needs to double exactly such that equal amounts can be passed into the two daughter cells during cell division.
DNA has two major functions in the cell:
• Replication, in dividing cells to from exact copies
• Carrying information (genetic code) for protein synthesis in all cells
Chromosomes must make copies of themselves so that when cells divide, each daughter cell must receive an exact copy of the genetic information. This copying of DNA is called DNA replication and takes place in a cell during interphase.
DNA replication occurs as follows:
- DNA double helix unwinds through activity of DNA helicase enzyme
- DNA helicase also breaks the hydrogen bonds holding complementary base pairs together and the two strands of the DNA molecule separate
- Both original DNA strands now act as templates for the synthesis of new DNA strands
- Free DNA nucleotides inside the nucleus align next to their complementary exposed bases
- DNA polymerase enzyme joins together the DNA nucleotides forming the sugar-phosphate backbone of the newly synthesised DNA strands
- The result is two new DNA molecules, each made up of one newly synthesised strand and one original strand that has been conserved from the original molecule
- This is called the semi-conservative hypothesis. This theory was proposed by Watson and Crick in 1954 and confirmed shortly after by the evidence provided by Meselson and Stahl’s experiments.
Experimental evidence for semi-conservative replication:
Experiments by Matthew Meselson and Franklin Stahl in 1958 to demonstrate the semi-conservative nature of DNA replication involved the use of an ultra-centrifuge. This rotates centrifuge tubes at very high speeds, with each centrifuge tube containing a liquid suspension (of caesium chloride into which the extracted DNA has been added). This results in the heavier or denser particles separating out at a lower point in the tube and the lighter or less dense particles separating out higher up the tube:
Experiments by Matthew Meselson and Franklin Stahl in 1958 to demonstrate the semi-conservative nature of DNA replication involved the use of an ultra-centrifuge. This rotates centrifuge tubes at very high speeds, with each centrifuge tube containing a liquid suspension (of caesium chloride into which the extracted DNA has been added). This results in the heavier or denser particles separating out at a lower point in the tube and the lighter or less dense particles separating out higher up the tube:
- The scientists cultured the bacterium, Escherichia coli, for several generations in a culture medium containing amino acids made with the heavy isotope of nitrogen 15N. The bacteria incorporated the 15N into their nucleotides and then into their DNA so that all the DNA contained 15N. They extracted the bacterial DNA and centrifuged it. The DNA settled at a low point in the tube (because all DNA molecules contained two strands both made from 15N)
- The 15N bacteria were washed, then transferred to a medium containing the normal, lighter form of nitrogen, 14N, and were allowed to divide once more
- When extracts of DNA from this first generation culture were centrifuged, the DNA was shown to have a mid-point density since each new DNA molecule contained one newly synthesised DNA strand containing 14N (the light isotope) and an original DNA strand containing N15 (the heavy isotope)
- When DNA extracts were taken from the second generation grown in 14N, the DNA settled at mid points and high points in the centrifuge tube. This was conclusive evidence for the semi-conservative hypothesis