DNA Notes - IB Biology



|SBI3U |DNA Structure (3.3 & 7.1) |

|The Chemistry of Life | |

Introduction

|Macromolecule |Monomer Unit |

|Carbohydrates |Monosaccharides |

|Proteins |Amino acids |

|Triglycerides |3 fatty acids and glycerol |

|DNA |Nucleotides |

|RNA |Nucleotides |

• There are 2 types of nucleic acids:

1) Deoxyribonucleic acid (DNA)

2) Ribonucleic acid (RNA)

• Both these nucleic acids are large macromolecules with molecular weights of 25,000 to over 50 billion atomic mass units (amu). They are linear, unbranched polymers that have repeating monomer units called NUCLEOTIDES.

• Nucleotides are composed of three parts:

1) A 5-carbon sugar

2) Phosphate group (PO43–)

3) Nitrogen base

• A combination of a 5-carbon sugar (pentose sugar) and a nitrogen base is called a NUCLEOSIDE (there is no phosphate group attached).

DNA VS. RNA

|DNA |RNA |

|Double stranded |Single stranded |

|Sugar = deoxyribose |Sugar = ribose |

|Bases = A, T, G, C |Bases = A, U, G, C |

|In eukaryotes, found in nucleus only |In eukaryotes, found in nucleus and cytoplasm |

|Storage of Genetic information |Involvement in Protein Synthesis: |

|Replication |Messenger (mRNA) |

|Transcription (template) |Transporter (tRNA) |

| |Associated with Ribosomes (rRNA) |

• In eukaryotes DNA is associated with proteins and is wound into chromosomes.

• In prokaryotes DNA is naked and is supercoiled into a series of small loops. In addition many bacteria have additional circular DNA called plasmids.

A comprehensive look at nucleotides and DNA

1) A 5-carbon sugar (pentose sugar because it has 5 carbons). A nucleotide contains either:

a) deoxyribose (found in DNA nucleotides)

b) ribose (found in RNA nucleotides)

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NOTE: The 2’C in deoxyribose has a second hydrogen instead of a hydroxyl group. Since it is missing an oxygen atom, it receives the prefix “deoxy.”

• These sugars are part of the backbone of DNA and they:

1) Attach to their phosphate group at 5’C,

2) Attach to their nitrogen base at 1’C, AND

3) Attach to the PO43– group of the nucleotide below at 3’C.

2) Phosphate group (PO43–):

• They are extremely polar covalent molecules.

• They form part of the DNA backbone, and they:

1) Attach to the 5’C of their pentose sugar, and

2) Attach to the 3’C of the nucleotide sugar above.

• A nucleotide can have either 1 phosphate group (mono), 2 phosphate groups (di), or 3 phosphate groups (tri).

o Example: adenosine triphosphate (ATP)

3) Nitrogen bases. These form the rungs of the DNA ladder:

• Unshared pair of electrons on the nitrogen atoms can acquire protons (hydrogen ions) ( the term base is used.

• They are hydrophobic molecules.

• There are 5 nitrogen bases: Adenine, Guanine, Cytosine, Thymine (used only in DNA) and Uracil (used only in RNA). These nitrogen bases attach to 1’C of the pentose sugar.

• There are 2 categories of nitrogen bases

I) Purines – These are nitrogen bases with a double ring that attach to the 1’C on the pentose sugar via 9'N on the second ring.

a) Adenine (A)

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b) Guanine (G)

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II) Pyrimidines – These are nitrogen bases with a single ring that attach to the 1’C on the pentose via the 1'N in the ring (bottom N).

1) Thymine (T)

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a) Cytosine (C)

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b) Uracil (U)

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• Thus, a nucleotide of adenosine triphoshate (ATP) is composed of adenine, ribose, and 3 phosphate groups.

• The DNA backbone is composed of repeating nucleotides (but the nitrogen base can differ).

• DNA is read from the 5’ ( 3’ direction so this strand is 5’ ATT GCG 3’.

Discovering the structure of DNA

• It was known that nucleic acids were composed of nucleotides (phosphate group, pentose sugar and nitrogen base) but how they were arranged was not determined until 1953 when a paper was published by James Watson and Francis Crick.

• Watson and Crick knew the research regarding DNA and 2 critical pieces were put together to reveal the structure of DNA:

1) Rosalind Franklin using X-ray crystallographic analysis of fibres of DNA (prepared by biochemist Maurice Wilkins) suggested that the structure of the DNA molecule was a helical coil, a spring-like spiral. The helix had a diameter of 2 nm (nanometres) and made a complete spiral every 3.4 nm.

2) Erwin Chargaff discovered that the number of guanine units equals the number of cytosine units and the number of adenine units equals the number of thymine units. This is known as Chargaff’s rule, along with the rule that pairings of a purine with a pyrimidine allows for the constant width of a DNA molecule.

• There are 4 nitrogen bases in DNA (adenine, thymine, cytosine and guanine) and using Franklin’s and Chargaff’s data Watson and Crick proposed that:

1) There are 2 hydrogen bonds that hold adenine and thymine together (2nm in diameter). This base pairing is found only in DNA. Note: In RNA the base uracil replaces the thymine.

• A-T has 2 hydrogen bonds (as does A-U).

2) There are 3 hydrogen bonds that hold cytosine and guanine together (2nm in diameter). This base pairing if found in both DNA and RNA.

• C-G has 3 hydrogen bonds.

• The arrangement of the more hydrophobic bases in the interior of the molecule and the more hydrophilic phosphates and sugars to the exterior made sense to Watson and Crick as DNA existed in an aqueous medium (nucleoplasm).

• A spiral turn of 3.4 nm (from Franklin’s work) allowed for 10 nitrogen base pairings.

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• Watson and Crick proposed that:

1) DNA was a double helix composed of two polynucleotide chains that are held together by hydrogen bonding between paired nitrogen bases. Each hydrogen bond is weak but collectively are very strong.

2) The nucleotides in the sugar-phosphate backbone are held together by covalent bonds called phosphodiester bonds between the phosphate of one nucleotide and the sugar of the next nucleotide.

3) The two sugar-phosphate backbones of the double helix are oriented in opposite directions, antiparallel to one another. Each polynucleotide chain has a nitrogen base sequence running in a 5'( 3' direction.

Organization of genetic material

• Eukaryotic cells contain more than 2m of DNA (over 6 billion base pairs).

• DNA is wound twice around 8 histone proteins with an additional 9th histone holding it all together. This is called a nucleosome.

• This nucleosome is then further wound into chromatin fibres.

• These nucleosomes help regulate transcription (needed to express genes) by permitting or restricting access to the protein-coding region of DNA.

DNA and genes

• Genes are sections of DNA that are located on chromatin/chromosomes that code for specific proteins. They are passed from parent to offspring.

• A region before the gene is called a promoter and it is like a switch that can turn a gene on.

o Example: After you eat the insulin gene in pancreatic cells will be told to make insulin molecules (insulin moves sugars into the cell).

• A region after the gene is called a terminator.

o Example: When you make enough insulin, it will turn the gene off.

• Eukaryotic genes contain sections of DNA referred to as introns and exons.

• Areas of DNA that do not code for genes are called introns (aka junk DNA). The function and importance of these areas is still being researched. They are like “bookends” to genes.

• Areas of DNA that code for genes are called exons.

• Highly repetitive DNA accounts for 5%–45% of the human genome. They usually contain 5–300 base pairs that can repeat up to 100,000 times. The function of these sequences is still being researched, but it has been determined that they are transposable (can move from one location to another within the genome).

• The human genome contains 30,000 genes and they are carried by 46 chromosomes. All the DNA in one nucleus of a single cell adds up to 2.5 billion (2,500,000,000) nucleotides long.

• The insulin gene has 51 amino acids and it takes 3 nucleotides to code for 1 amino acid (more on this later), therefore it takes 153 nucleotides to code for an insulin molecule.

|SBI3U |DNA Replication (3.4 & 7.2) |

|The Chemistry of Life | |

The Meselsono-Stahl experiment

• DNA is unique among macromolecules – it can replicate itself.

• In 1958, Matthew Meselson and Franklin W. Stahl demonstrated that DNA replication was semiconservative.

o A semiconservative model of DNA replication was predicted by the Watson & Crick model of DNA.

• Meselson and Stahl grew E. coli bacteria in a growth medium containing 15N, a heavy isotope.

o The bacteria reproduced and in time it was determined that the bacteria had incorporated the 15N into their DNA.

• Meselson and Stahl transferred the E. coli to a medium of 14N.

o After 1 generation, the bacteria had DNA of intermediate density.

o After 2 generations, 50% of the bacteria had DNA of intermediate density and 50% of the bacteria had DNA of 14N density

o After 3 generations, 25% of the bacteria had DNA of intermediate density and 75% of the bacteria had DNA of 14N density

A step-by-step summary of DNA replication

1) There are promoter regions on DNA that indicate the spot or spots where DNA replication will initiate – origin(s) of replication – designating the beginning of a gene. Prokaryotic cells have one origin of replication. Eukaryotic cells have many origins of replication.

2) To separate the 2 polynucleotide strands of DNA, 3 types of enzymes are involved:

a. Topoisomerases: relieve molecular stress and untangle snarls in DNA by breaking them and resealing the strand.

b. Helicases: unwind the double-helix by breaking the hydrogen bonds between complementary pairs of nitrogen bases (similar to unzipping a zipper).

c. Single-stranded binding proteins: help keep the separated strands of DNA from re-annealing.

3) A RNA primer (usually about 5 RNA nucleotides long) is added by RNA primase (an enzyme) to the 3’ end of both strands of DNA to initiate the replication process.

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4) As the hydrogen bonds between nucleotide pairs are broken, free floating DNA nucleotides (deoxynucleotide triphosphates) that are present in the nucleoplasm are carried by DNA polymerase III to be bonded to the DNA template.

PROBLEM #1: It was discovered that the 2 strands of DNA do not replicate in the same manner.

5) One strand (the 5’ ( 3’) simply adds nucleotides to the 3’ end. It grows in the 5’ ( 3’ direction by adding nucleotides found in the nucleus, growing towards the Y-junction. This strand is called the LEADING strand.

6) The other strand (the 3’ ( 5’) is called the LAGGING strand. DNA can only be read in the 5’ ( 3’ direction, so the lagging strand cannot start immediately – it has to wait until a section of DNA has been separated. Thus, a new primer is added by RNA primase allowing the exposed section of DNA to by copied by DNA polymerase III in the 5’ ( 3’ direction. Eventually another section of DNA on the lagging strand becomes exposed, a new primer is added by RNA primase, and the DNA is copied. This process is repeated until the lagging strand is fully copied, creating short sections of DNA called Okazaki fragments.

PROBLEM #2: The Okazaki fragments are not connected into a continuous strand, and the primer is not DNA – it is RNA.

7) DNA polymerase I removes the RNA primer and replaces them with DNA nucleotides.

8) DNA ligase (a linking enzyme) helps to connect the Okazaki fragments, creating a continuous strand of DNA on the lagging strand.

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DNA proofreading

• The high degree of accuracy in the process of DNA replication is not solely the result of specific base pairing (A-T, C-G).

• Base pairing errors occur about every 1 in 10,000 pairings.

o Human cells pair bases at a rate of 50 base pairings per second.

o Bacteria pair bases at a rate of 500 base pairings per second.

o It is very evident that 1 erroneous base pairing every 10,000 could be costly causing numerous mutations within a species.

• BUT!!!... Only 1 in a billion nucleotides is incorrectly paired.

• In bacteria, DNA polymerases check the DNA sequence for base pairing errors.

o When an incorrect pairing is found, the DNA polymerases remove the incorrect nucleotide and replace it with the correct one.

• It is not known if eukaryotic cells have this function, but a similar mechanism exists.

• Scientists have identified 50 different types of DNA repair enzymes.

o This system of repair enzymes can repair damage to the DNA molecule caused by:

▪ Reactive chemicals (benzene, toluene, DDT, etc.).

▪ Radioactive emissions.

▪ X-rays.

▪ Ultra-violet (UV) radiation exposure.

o These help to maintain the integrity of the DNA nucleotide sequence.

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