nucleic acid and dna replication

structure of nucleotides

• nucleic acids are a class of biomolecules, and there are two types of nucleic acids:

  • DNA ⇒ deoxyribonucleic acid

  • RNA ⇒ ribonucleic acid

• nucleic acids are polymers made from monomers known as nucleotides

• a nucleotide is made up of 3 components: a phosphate group (gives acidic character and negative charge), a pentose sugar (5 carbon sugar and a nitrogenous base)

• formed via a condensation reaction ⇒ removes two water molecules

  • pentose sugar is bonded to a nitrogenous base at carbon 1

  • phosphate group is bonded to the pentose sugar molecule at carbon 5

• DNA and RNA differ in the type of pentose they contain

  • ribose (RNA): carbon 2 is attached to a hydroxyl group (-OH)

  • deoxyribose (DNA): carbon 2 is attached to a hydrogen atom → due to absence of OH group at carbon 2 in deoxyribose sugar, DNA is less reactive than RNA

• there are 5 different types of nitrogenous bases found in nucleic acids → classified as purines or pyrimidines

  1. purines → 2 rings

     • adenine (A), guanine (G)

  2. pyrimidines → 1 ring

     • thymine (T) [in DNA only], cytosine (C), uracil (U) [in RNA only]

structure and formation of polynucleotides

• nucleic acids are formed by joining nucleotides together

  • -OH group on carbon 3 of pentose sugar of a nucleotide

  • phosphate group on carbon 5 of pentose sugar of an adjacent nucleotide

  • via condensation reaction

  • bond formed ⇒ phosphodiester bond

• addition of many nucleotides produces a long polynucleotide chain/strand with a backbone of alternating sugar and phosphate groups and bases projecting sideways from the sugars

• the two ends of a polynucleotide chain are different from each other ⇒ polynucleotide chains have directionality along its sugar phosphate backbone, from 5’ to 3’

  • 5’ end: a free phosphate attached to a carbon 5 of a pentose sugar

  • 3’ end: a free hydroxyl group on a carbon 3 of a pentose sugar

• each polynucleotide strand consists of only 1 type of monomer→ DNA or RNA

structure of DNA

• each DNA molecule is made up of 2 strands of polynucleotides

  • each polynucleotide strand is made up of deoxyribonucleotides joined by phosphodiester bonds

• 2 strands of polynucleotides are wound to form a double helix

  • two strands are anti-parallel (i.e. opposite directions) to each other ⇒ 5’→3’ direction for one strand, 3’→5’ direction for the other strand

  • one complete turn of DNA double helix consists of 10 base pairs → distance of 3.4nm

  • sugar-phosphate backbone of each strand is on the exterior of the helix, while the nitrogenous bases are paired in the interior of the helix

  • the winding of the molecule creates major and minor grooves

• DNA molecule has a uniform width of 2 nm ⇒ the width of a base pair, consisting of the width of a purine and a pyrimidine

  • hydrogen bonds between complementary bases hold the two chains together

    • 2 hydrogen bonds between Adenine and Thymine

    • 3 hydrogen bonds between Cytosine and Guanine

  • since the two strands are complementary to each other, the ratio is

    • A : T = 1 : 1

    • G : C = 1 : 1

    • Purines (A+G) : Pyrimidines (T+C) = 1:1

• hydrophobic interactions between stacked nitrogenous bases stabilises the double helix

structure-function relationship of DNA

1. DNA function as genetic material that is passed from one generation of cells to the next (hereditary material), the structure of DNA must allow for:

   • accurate DNA replication and DNA repair

     structure	function
     double helix consisting of 2 strands	each DNA parental strand acts as a template for the • synthesis of the daughter strand ⇒ ensures accurate replication so that the daughter cells have identical DNA molecules as the parental cell • proofreading and repair of damaged strand if mutation occurs, so that the DNA sequence can be maintained

   • stability of hereditary material to remain unchanged

     structure	function
     antiparallel strands	allows many hydrogen bonds to form between complementary bases, and hold the two polynucleotide chains together
     complementary base pairing between purines and pyrimidines	• hold two polynucleotide chains together • ensure a constant width of 2.0nm maintaining a stable structure • stable structure maintains DNA sequence throughout the lifespan of cell • allow each strand to act as a template during DNA replication → (a)(i)
     stacked nitrogenous bases	hydrophobic interactions formed between stacked bases stabilises structure of double helix
     deoxyribose sugar in DNA nucleotide	less chemically reactive and more resistant to hydrolysis. ⇒ DNA sequence is maintained
     phosphodiester bonds between adjacent nucleotides within each strand	strong covalent bonds stabilise structure of the double helix

2. DNA also functions to store genetic information for gene expression

   structure	function
   sequence of bases forms genes	• code for functional gene products (e.g. polypeptide, tRNA, rRNA) • either strand acts as a template for the synthesis of RNA via complementary base pairing
   major and minor grooves formed from winding of DNA molecule	major grooves allow for binding of proteins (transcription factors) that regulate gene expression

semi-conservative DNA replication

process by which a double-stranded DNA molecule is copied to produce two identical DNA molecules

• sequence of the bases is the same

• [FAQ] semi-conservative because:

  • after hydrogen bonds between bases break and 2 parental strands separate,

  • each parental DNA strand is used as template to synthesize a new daughter strand

  • each new DNA molecule is a hybrid of 1 parental strand and 1 daughter strand

• before mitosis or meiosis, the doubling of DNA content needs to occur during S phase of interphase in eukaryotes

  • mitosis: the doubling ensures that daughter cells has identical copies of DNA and thus are genetically identical to the parental cells

before DNA replication begins → during G1 phase of interphase

• the following materials are imported from the cytoplasm into the nucleus via the nuclear pores

  • free deoxyribonucleotides ⇒ provided as nucleoside triphosphates e.g. ATP, GTP, CTP, TTP → these are also known as activated nucleotides (bases attached to phosphate groups)

  • free ribonucleotides ⇒ to form RNA primers

  • enzymes → helicase, primase, DNA polymerase, ligase

  • ATP

• DNA replication begins at sites on the DNA known as origin of replication

  • prokaryotes: one origin of replication

  • eukaryotes: many origins of replication

1. separating the double helix → dna replication

1. helicase binds to DNA molecule at the origin of replication, and disrupts hydrogen bonds between complementary bases

2. helicase separates/unzips parental strands

3. single-strand binding proteins stabilize the unwound helix and prevent rewinding of double helix

4. a replication bubble with two Y-shaped replication forks are formed

   • replication proceeds in BOTH directions (bidirectional) from the origin until the entire DNA molecule is replicated

2. synthesis of RNA primers → dna replication

5. primase synthesises RNA primer using free ribonucleotides (RNA) in the 5’→3’ direction [DNA template is read in the 3’ to 5’ direction]

   • occurs via complementary base pairing with the parental strand which acts as a template

[FAQ] why are primers needed for DNA synthesis?

• DNA polymerase cannot initiate DNA synthesis but can only add deoxyribonucleotide to free 3’-OH end of an
  existing strand in 5’→3’ direction

• this is because shape of DNA polymerase active site is complementary to shape of 5’ phosphate group of in-coming nucleotide and 3’-OH of the last nucleotide of growing daughter strand

• therefore, primers (short chains of ribonucleotides) provide free 3’-OH end for DNA polymerase to add deoxyribonucleotides

3. synthesis of daughter DNA strands → dna replication

6. DNA polymerase reads template strand in the 3’→5’ direction and synthesizes daughter strand in the 5’→3’ direction

   • free deoxyribonucleotides complementary base pairs with template strand:

     1. adenine base pairs with thymine, and vice versa (A=T)

     2. cytosine base pairs with guanine, and vice versa (C≡G)

   • phosphodiester bonds are formed between adjacent deoxyribonucleotides via condensation reactions

   • DNA polymerase also proofreads as it synthesises the daughter strand

     • if a nucleotide in the daughter strand is wrongly paired with template, the DNA polymerase will remove and replace with correct nucleotide

4. leading and lagging strands → dna replication

7. since the 2 parental strands are anti-parallel, the 2 daughter strands (leading and lagging strands) are synthesized in the opposite direction by DNA polymerases

   • leading strand is synthesised continuously towards the replication fork

     • only one primer is needed at the origin of replication

     • DNA polymerase adds new nucleotides in the 5’ →3’ direction without any breaks

   • lagging strand is synthesised discontinuously as Okazaki fragments away from the replication fork

     • as helicase separates DNA strands at the replication fork to expose the DNA templates, new primers will be synthesised by primase

     • DNA polymerase will add free nucleotides to the primer in the 5’ → 3’ direction, thus forming the Okazaki fragment

the overall direction of replication is still towards the replication fork as helicase separates the double-stranded DNA molecule

[FAQ] why is one daughter strand synthesized continuously while the other synthesized discontinuously?

1. the two parental strands are anti-parallel

2. synthesis of daughter DNA strands always starts at the origin of replication

3. DNA polymerase can only add deoxyribonucleotides to free 3’-OH end of an
   existing strand in 5’→3’ direction

4. therefore, DNA polymerase can only synthesise daughter strand in the 5’ to 3’ direction

5. leading strand is synthesized towards the replication fork

6. lagging strand is synthesized as Okazaki fragments away from replication fork
   before they are joined together by phosphodiester bonds

5. replacement of RNA primers with DNA → dna replication

1. another DNA polymerase replaces the RNA primers with deoxyribonucleotides

2. DNA ligase seals the gaps (nicks) between the Okazaki fragments by catalysing the formation of phosphodiester bonds between them

3. at the end, two DNA molecules are formed

   • each DNA molecule consists of 1 parental strand and 1 daughter strand, which will wind to form a double helix → semi-conservative

after DNA replication

• after DNA replication, proteins (histones) associate with the DNA and package to form chromatin

  • through multiple levels of packing, DNA can fit into the small space in the nucleus

process of dna replication for both prokaryotes and eukaryotes

• process of DNA replication is generally the same for both prokaryotes and eukaryotes

  	prokaryotes	eukaryotes
  when DNA replication occurs	prior to binary fission	S phase of interphase
  location	cytosol	nucleus
  DNA molecule structure	single circular	multiple and linear

evidence for semi-conservative replication

1. generation zero cells:

   • 100% 15N-15N DNA

2. first generation cells:

   • DNA from first generation cells (grown in 14N medium) forms one band between pure 15N-15N
     DNA and pure 14N-14N DNA (14N-14N) in caesium chloride

   • it supported semi-conservative replication → all DNA (100%) of the “first generation cells” was hybrid ⇒ one 14N strand and one 15N strand

3. second generation cells:

   • half (50%) was hybrid 14N-15N DNA, half (50%) was pure 14N-14N DNA

   • results confirmed semi-conservative model

• as bacteria continue to grow in the 14N-containing medium, DNA band with 14N-14N DNA molecules will be thicker, because the percentage of 14N-14N DNA molecules keep increasing in
  subsequent generations

• percentage of 14N-15N DNA molecules decreases in subsequent generations, this hybrid DNA molecules will always be present due to semi-conservative replication hypothesis

end replication problem

• limitations in the DNA polymerase create problems for linear DNA in eukaryotes

  • DNA polymerase can only add free deoxyribonucleotides to an existing 3’ –OH group

what is the end replication problem

• it is the shortening of DNA molecule after each round of DNA replication

• this is due to a gap at the 5’ end of the daughter strand after removal of RNA primer located at the 5’ end of daughter strand

• the RNA primer cannot be replaced with DNA nucleotides because there is no existing 3’–OH group available [since is the end of the strand] for DNA polymerase to add
  deoxynucleotides

• as DNA polymerase can only add deoxyribonucleotides to an existing 3’ –OH group