Understanding Nucleic Acids and DNA Processes

Instructions Do not edit the instructions below. Start your presentation on the tabs on the left according to your allocation. Please add in any drawing or images if necessary to aid your explanation. NO copying of lecture notes! Remember to be CONCISE and show your learning! 2.1 Content Allocation: 1) Nucleic acids monomer structure (as a biomolecule, DNA and RNA) (a) Components (b) How to draw (c) How to tell 5' and 3'? 2) Structure of polynucleotides DNA (Double Helix, Chargaff's rule) vs RNA (mRNA, tRNA, rRNA) 3) What are the 3 proposed models of DNA replication and How does the Meselson & Stahl experiment show us which is the correct one? 4) Brief Steps of the DNA Replication process - what are the main 6 proteins involved and what do they do? 5) Leading strand and lagging strand - why do these occur and what are the Okazaki fragments? How is DNA replication completed? 6) End Replication Problem: why do they occur and how does the cell protect itself against it? Learning Outcomes Candidates should be able to: (a) describe the structure and roles of DNA and RNA (tRNA, rRNA and mRNA) (knowledge of the structure and roles of mitochondrial DNA and chloroplast DNA is not required) (b) describe the process of DNA replication and how the end replication problem arises Nucleic acid monomer Nucleic acids (made up of nucleotides to form polynucleotides) - genetic material for all living organisms from dna to rna: an RNA copy (transcript) of the gene must first be made. This type of RNA is called a messenger RNA(mRNA), as it serves as a messenger between DNA and the ribosomes, molecular machines that read mRNA sequences and use them to build proteins. This progression from DNA to RNA to protein is called the “central dogma” of molecular biology. Deoxyribonucleic acid vs ribonucleic acid DNA * 2 strands of polynucleotide chains twisted to form double helix (anti parallel) * Carries genetic information of an organism RNA (formed via transcription of DNA) * only 1 polynucleotide chain * mRNA (messenger), tRNA (transfer), rRNA (ribosomal) * tRNA and rRNA are non -coding * mRNA carries genetic information for the synthesis of proteins after transcription Structure of nucleotides Consists of 1: Pentose sugar * DNA and RNA nucleotides differ in the type of pentose they contain: * in DNA, the pentose sugar is deoxyribose which has a H atom at C2 * in RNA, the pentose sugar is ribose which has an OH group at C2 2: Nitrogenous base nitrogenous bases are categorised into purines and pyrimidines according to the number of rings: - purines (2 rings) - 2 types: adenine (A) and guanine (G) - pyrimidines (1 ring) - 3 types: cytosine (C), uracil (U) and thymine (T) * adenine, cytosine and guanine (A,T,G) can be found in both DNA and RNA nucleotides - thymine(T) in DNA nucleotides only and uracil(U) in RNA nucleotides only * the nitrogenous base is attached to C1 of the pentose sugar 3. Phosphate group * the phosphate group is attached to C5 of the pentose sugar * depending on the number of phosphate groups present, 3 types of nucleotide monomers are possible: (a) nucleoside monophosphate - 1 phosphate group (b) nucleoside diphosphate - 2 phosphate groups (c) nucleoside triphosphate - 3 phosphate groups * nucleoside triphosphates are the precursors to nucleic acid synthesis of both DNA and RNA. The release of 2 terminal phosphate groups provides the energy for the polymerisation process of joining the nucleotide monomers together Formation of nucleotide: 1. The 3 components are linked up by 2 condensation reactions 2. The 1st condensation reaction links covalently C1 of the pentose sugar to the nitrogenous base, giving a nucleoside 3. the 2nd condensation reaction links covalently C5 of the pentose sugar to the phosphate group, giving a nucleoside monophosphate * 1 water molecule is removed for each condensation reaction; this means a net removal of 2 water molecules for each nucleotide formed. Polynucleotide chains A consequence of the structure of nucleotides is that a polynucleotide chain has directionality – that is, it has two ends that are different from each other. At the 5’ end, or beginning, of the chain, the 5’ phosphate group of the first nucleotide in the chain sticks out. At the other end, called the 3’ end, the 3’ hydroxyl of the last nucleotide added to the chain is exposed. DNA sequences are usually written in the 5' to 3' direction, meaning that the nucleotide at the 5' end comes first and the nucleotide at the 3' end comes last. As new nucleotides are added to a strand of DNA or RNA, the strand grows at its 3’ end, with the 5′ phosphate of an incoming nucleotide attaching to the hydroxyl group at the 3’ end of the chain. This makes a chain with each sugar joined to its neighbors by a set of bonds called a phosphodiester linkage. Drawing: C1: where nitrogenous base attaches C2: to tell if its DNA or RNA C5: where phosphate group attaches Attachment of nitrogenous base and phosphate group are both condensation reactions Nitrogenous base is attached before phosphate group Nitrogenous bases😟😟: Phosphate group: Deoxyribonucleic acid, or DNA, chains are typically found in a double helix, a structure in which two matching (complementary) chains are stuck together, as shown in the diagram at left. The sugars and phosphates lie on the outside of the helix, forming the backbone of the DNA; this portion of the molecule is sometimes called the sugar-phosphate backbone. The nitrogenous bases extend into the interior, like the steps of a staircase, in pairs; the bases of a pair are bound to each other by hydrogen bonds. The two strands of the helix run in opposite directions, meaning that the 5′ end of one strand is paired up with the 3′ end of its matching strand. (This is referred to as antiparallel orientation and is important for the copying of DNA.) Because of the sizes and functional groups of the bases, base pairing is highly specific: A can only pair with T, and G can only pair with C, as shown below. This means that the two strands of a DNA double helix have a very predictable relationship to each other. When 2 DNA sequences match such that they can stick to each other in an antiparallel fashion and form a helix, they are said to be complementary. The 5' end has a phosphate group attached to the 5th carbon, while the 3' end has a hydroxyl group attached to the 3rd carbon. Structures of nucleic acid polymers Nucleic acids (polynucleotides) * Polynucleotides are formed by the polymerisation (or adding together) of nucleotide monomers (either deoxyribonucleotides or ribonucleotides) are added together * A dinucleotide is formed when a condensation reaction occurs between a hydroxyl group (–OH group) at C3 of the pentose sugar on 1 nucleotide and –OH group of the phosphate attached to C5 of the pentose sugar on another nucleotide * The linkage that connects two nucleotides via a phosphate group is a phosphodiester linkage * Continual addition of nucleotides produces a polynucleotide strand, with alternating pentose sugars and phosphate groups Structure of polynucleotides A polynucleotide strand has a sugar-phosphate backbone and nitrogenous bases projecting sideways / outwards: i. Sugar-phosphate backbone - Made up of alternating pentose sugars and phosphate groups, linked by phosphodiester linkages - All the pentose sugars and phosphate groups along this sugar-phosphate backbone are identical. ii. Nitrogenous bases project sideways / outwards from the pentose sugars - Because the nitrogenous bases at each nucleotide can vary, this gives rise to a specific sequence of nitrogenous bases (A,T,C,G) along the length of the polynucleotide strand; in the case of DNA, it is this unique sequence of nitrogenous bases that carries genetic information for the production of gene product such as proteins, which ultimately influences an organism’s appearance, behavior and development. A polynucleotide strand has a 5’ end and a 3’ end: - 5’ end refers to the end with a phosphate group attached to C5 of the pentose sugar. - 3’ end refers to the other/opposite end with a free hydroxyl group attached to C3 of the pentose sugar. - During replication or transcription, the genetic information along the polynucleotide strand is read in the 5’ → 3’ direction - The strong covalent phosphodiester linkages between adjacent nucleotides along the sugar- phosphate backbone confers strength and stability to the polynucleotide strand and prevents it from breaking. Polynucleotides: * Polymerisation of nucleotide monomers (also condensation reaction) * Phosphate group from another nucleotide is attached to C3 on first nucleotide DNA (Double Helix, Chargaff's Rule) * Structure: DNA is a double-stranded molecule where two polynucleotide chains are coiled around each other to form a double helix. * Sugar: Deoxyribose. * Bases: Adenine (A), guanine (G), cytosine (C), and thymine (T). * Chargaff's Rule: The amount of adenine (A) is equal to the amount of thymine (T), and the amount of guanine (G) is equal to the amount of cytosine (C). * Base Pairing: A pairs with T, and G pairs with C. * Double Helix: The two strands are held together by hydrogen bonds between the bases, and the helix is right-handed RNA (mRNA, tRNA, rRNA) * Structure: RNA is usually single-stranded, but it can fold into complex 3D shapes. * Sugar: Ribose. —> has OH * Bases: Adenine (A), guanine (G), cytosine (C), and uracil (U). * Types: * mRNA (messenger RNA): Carries the genetic code from DNA to the ribosomes for protein synthesis. * tRNA (transfer RNA): Transports amino acids to the ribosomes during protein synthesis. (Clover leaf- shaped) * rRNA (ribosomal RNA): A component of ribosomes, which are responsible for protein synthesis. Meselson Stahl Experiments 3 Proposed models of DNA replication 1. Dispersive replication: (Old and new segments dispersed around to form daughter DNA molecules) * Original parental DNA molecule breaks up into short segments * Short segments are used as templates for the formation of 2 new daughter DNA molecules * Segments are then joined together such that the newly synthesised daughter DNA molecules would contain a mixture of old and newly synthesised segments. 2. Conservative replication: (Original parental DNA molecule is conserved) * The double-stranded, double helix DNA molecule (original parental DNA molecule) acts as a template for an entirely new double helix DNA molecule to be formed. * The parent DNA molecule is intact and goes into one of the two daughter cells. The newly formed DNA double helix molecule goes into another daughter cell. 3. Semi-conservative replication: (Both strands of original parental DNA molecule is conserved separately) * The 2 strands on the DNA double helix molecule separate when the hydrogen bonds between the base pairs are disrupted. * Each parental DNA strand then acts as a template for the formation of one new DNA strand. Hydrogen bonds form between bases of the original, parental strand and a newly synthesised DNA strand to form the daughter DNA molecule. * From 1 original parental DNA molecule, it will replicate to form 2 newly synthesised daughter DNA molecules. Each daughter DNA molecule is a hybrid, consisting of one original, parental strand and one newly synthesised, daughter strand. Messelson and Stahl experiment supports Semi-conservative replication model Mode of DNA replication: Meselson-Stahl experiment (article) | Khan Academy DNA Replication process There are 6 proteins involved in the steps of replication each with specific roles * Helicase * Single-stranded DNA binding proteins * Enzyme primase * DNA Polymerase (I and III) * DNA ligase Helicase helps with the initial parts of replication . In DNA the specific sites where the origin of replication occurs (ori ) has a specific sequence of nucleotides with a high ratio of it being adenine and thymine . In this case Helicase will recognise and bind to the ori , where ist breaks the hydrogen bonds between the two strands of complementary bases which form the DNA helix , causing the DNA molecule to unwind into two separate strands.(Helicase has a complementary binding site to the sequence of dna molecule) This unwinding will result in a replication bubble with 2 Y shape regions known as replication forks that move apart in opposite directions. Note: Unwind and unzip are different * Unzip: involves bond breaking between nitrogenous bases After the unwinding and unzipping happens Single-stranded DNA binding proteins will attach itself along the two unpaired strands to keep the strands from reforming bonds with one another. These two separate strands are base templates for the synthesis of 2 new complementary DNA strands with new sequences These binding proteins will stay until new DNA strands are formed They stay attached to the single DNA strands until DNA polymerase comes along and starts building the new complementary strand. Enzyme primase will synthesizes a short RNA primer on the DNA template strand during replication. This primer provides a starting point for DNA polymerase to begin addition of the nucleotides. An RNA primer is a short strand of RNA (usually about 5–10 nucleotides long) that provides a starting point for DNA synthesis. During DNA replication, enzymes like DNA polymerase cannot begin making a new DNA strand on their own and they can only add nucleotides to an existing strand. * RNA polymerase(unlike DNA polymerase): Can add nucleotides without pre-existing chain during transcription Specifically the enzymes can only add deoxyribonucleotides to free 3’OH end of a pre-existing polynucleotide chain that has already base-paired with the template because it does not act as a ribosome and cannot start creating its own strands. DNA polymerase can’t add nucleotides to single-stranded DNA because it needs a free 3’-OH group (hydroxyl group) from an existing nucleotide strand to attach the next nucleotide. The reason why a primer is required because DNA synthesis is a chemical reaction where the 3’-OH of the last nucleotide attaches to the phosphate group of the incoming nucleotide. Without that 3’-OH, the reaction can’t start. When DNA polymerase moves along the strand and adds new nucleotides, it pushes off the binding proteins because they are no longer needed and the new double-stranded structure is now stable on its own. Two enzymes will aid in the addition of deoxyribonucleotides and the formation of phosphodiester bonds (A phosphodiester bond is the type of bond that links the nucleotides together in DNA and RNA strands joining one sugar to the phosphate group of complementary bases) between the nucleotides (complementary bases) which are polymerase III and polymerase I . Extra:Polymerase II is for DNA repair and not involved in replication Polymerase III is responsible for the addition of the nucleotides , elongating the strand while polymerase I removes primers from DNA strand and replaces it with nucleotides DNA ligase forms phosphodiester linkages between newly synthesized DNA fragments in the strand to form a continuous strand of DNA. Leading, lagging & Okazaki Leading and Lagging strands Leading Strand: * Direction: The leading strand is synthesized in the 5' to 3' direction, towards the replication fault. * How it's made: Since DNA polymerase can only add nucleotides to the 3' end of an existing strand (the 3' to 5' direction), the strand that is oriented 3' to 5' can be synthesized continuously. The leading strand is synthesized continuously , as the replication fork opens up and DNA polymerase follows it, continuously adding nucleotides in the correct direction. Lagging Strand: * Direction: The lagging strand runs in the 5' to 3' direction (opposite to the direction of the replication fork). * How it's made: This causes a problem because DNA polymerase can’t add nucleotides in the 5' to 3' direction of the parental strand (since it can only add in the 3' to 5' direction). So, the lagging strand is made in short segments, known as Okazaki fragments. DNA polymerase adds nucleotides to the 3' end of these Okazaki fragments, but once one fragment is finished, the enzyme starts again at a different spot. This creates multiple short segments, which are later connected by an enzyme called DNA ligase to form a continuous strand. * Okazaki fragments: Okazaki fragments are short segments of DNA that are synthesized on the lagging strand during DNA replication. Since DNA can only be synthesized in the 5' to 3' direction, and the lagging strand runs in the 3' to 5' direction, the synthesis on the lagging strand happens in a bit of a "backward" manner. Instead of being made continuously (like on the leading strand), the lagging strand is made in short, discontinuous segments. These segments are the Okazaki fragments. Key Differences: * Leading Strand: Continuous synthesis in the direction of the replication fork. * Lagging Strand: Discontinuous synthesis, made in fragments, opposite to the direction of the replication fork. * Analogy: -“unzipping” a zipper—one side (the leading strand) just unzips and follows directly. The other side (the lagging strand) needs to start from multiple points along the zipper and work backwards, creating smaller sections that are later stitched together. Mr Leong’s wisdom can’t be found online: Leading and lagging strands are synthesized in opposite directions as the DNA strands are antiparallel and are opposite directions. Lagging strand has to wait for more of the parental strand to be unzipped for synthesis. Leading strand can be synthesized as parental strand is unzipped. Typical questions: Why is one discontinuous? Why is one continuous? Why is the discontinuous strand formed dim fragments? End Replication Problem End Replication Problem: End Replication Problem is the problem where DNA daughter strands after replication are shorter than the parental strands due to the inability to attach DNA nucleotides after the removal of the RNA primer. Why it occurs: Nucleotides can only be attached to the daughter strand at the OH of the 3’ ends At the extreme 5’ ends of the lagging strand, when the RNA primer is removed by DNA polymerase I, there is no 3’ end available to attach nucleotides to, hence the daughter strand is left shorter than the parental strand. Over multiple replications, the daughter strands will get shorter and shorter, which will eventually result in genetic information to be lost. How the cell protects itself against this: 1. Telomeres 2. Telomerase Telomeres are a sequence of nucleotides (5’ -TTAGGG- 3’)at the ends of the DNA strands that do not code for anything and are repetitive. It serves as a sort of buffer to delay the erosion of nucleotides which code for important genetic information. (does not solve problem) Telomerase is an ribonucleoprotein that helps to lengthen telomeres. It consists of: * Telomerase RNA which provides a template to guide synthesis of telomere sequences * Telomerase reverse transcriptase which catalyses the synthesis of DNA from RNA

NO copying of lecture notes! Remember to be CONCISE and show your learning!

2.1 Content Allocation:

1) Nucleic acids monomer structure (as a biomolecule, DNA and RNA)
(a) Components (b) How to draw (c) How to tell 5' and 3'?

2) Structure of polynucleotides DNA (Double Helix, Chargaff's rule) vs RNA (mRNA, tRNA, rRNA)

3) What are the 3 proposed models of DNA replication and How does the Meselson & Stahl experiment show us which is the correct one?

4) Brief Steps of the DNA Replication process - what are the main 6 proteins involved and what do they do?

5) Leading strand and lagging strand - why do these occur and what are the Okazaki fragments? How is DNA replication completed?

6) End Replication Problem: why do they occur and how does the cell protect itself against it?

Learning Outcomes
Candidates should be able to:

(a)         describe the structure and roles of DNA and RNA (tRNA, rRNA and mRNA) (knowledge of the structure and roles of mitochondrial DNA and chloroplast DNA is not required)

(b) describe the process of DNA replication and how the end replication problem arises
Nucleic acid monomer
Nucleic acids (made up of nucleotides to form polynucleotides) - genetic material for all living organisms

from dna to rna: an RNA copy (transcript) of the gene must first be made. This type of RNA is called a messenger RNA(mRNA), as it serves as a messenger between DNA and the ribosomes, molecular machines that read mRNA sequences and use them to build proteins. This progression from DNA to RNA to protein is called the “central dogma” of molecular biology.

Deoxyribonucleic acid vs ribonucleic acid
DNA      
* 2 strands of polynucleotide chains twisted to form double helix (anti parallel)
* Carries genetic information of an organism
RNA (formed via transcription of DNA)
*  only 1 polynucleotide chain
* mRNA (messenger), tRNA (transfer), rRNA (ribosomal) 
* tRNA and rRNA are non -coding
* mRNA carries genetic information for the synthesis of proteins after transcription

Structure of nucleotides

Consists of 1: Pentose sugar
* DNA and RNA nucleotides differ in the type of pentose they contain:
*  in DNA, the pentose sugar is deoxyribose which has a H atom at C2
*  in RNA, the pentose sugar is ribose which has an OH group at C2

2: Nitrogenous base
nitrogenous bases are categorised into purines and pyrimidines according to the number of rings: 
- purines (2 rings) - 2 types: adenine (A) and guanine (G)
- pyrimidines (1 ring) - 3 types: cytosine (C), uracil (U) and thymine (T) 
* adenine, cytosine and guanine (A,T,G) can be found in both DNA and RNA nucleotides - thymine(T) in DNA nucleotides only and uracil(U) in RNA nucleotides only
* the nitrogenous base is attached to C1 of the pentose sugar

3. Phosphate group
* the phosphate group is attached to C5 of the pentose sugar
* depending on the number of phosphate groups present, 3 types of nucleotide monomers are possible:
(a) nucleoside monophosphate - 1 phosphate group
(b) nucleoside diphosphate - 2 phosphate groups
(c) nucleoside triphosphate - 3 phosphate groups
* nucleoside triphosphates are the precursors to nucleic acid synthesis of both DNA and RNA. The release of 2 terminal phosphate groups provides the energy for the polymerisation process of joining the nucleotide monomers together

Formation of nucleotide: 
1. The 3 components are linked up by 2 condensation reactions 
2. The 1st condensation reaction links covalently C1 of the pentose sugar to the nitrogenous base, giving a nucleoside
3. the 2nd condensation reaction links covalently C5 of the pentose sugar to the phosphate group, giving a nucleoside monophosphate
* 1 water molecule is removed for each condensation reaction; this means a net removal of 2 water molecules for each nucleotide formed.
Polynucleotide chains
A consequence of the structure of nucleotides is that a polynucleotide chain has directionality – that is, it has two ends that are different from each other. At the 5’ end, or beginning, of the chain, the 5’ phosphate group of the first nucleotide in the chain sticks out. At the other end, called the 3’ end, the 3’ hydroxyl of the last nucleotide added to the chain is exposed. DNA sequences are usually written in the 5' to 3' direction, meaning that the nucleotide at the 5' end comes first and the nucleotide at the 3' end comes last.
As new nucleotides are added to a strand of DNA or RNA, the strand grows at its 3’ end, with the 5′ phosphate of an incoming nucleotide attaching to the hydroxyl group at the 3’ end of the chain. This makes a chain with each sugar joined to its neighbors by a set of bonds called a phosphodiester linkage.
Drawing:

C1: where nitrogenous base attaches 
C2: to tell if its DNA or RNA
C5: where phosphate group attaches

Attachment of nitrogenous base and phosphate group are both condensation reactions
Nitrogenous base is attached before phosphate group

Nitrogenous bases😟😟:                                                Phosphate group:

Deoxyribonucleic acid, or DNA, chains are typically found in a double helix, a structure in which two matching (complementary) chains are stuck together, as shown in the diagram at left. The sugars and phosphates lie on the outside of the helix, forming the backbone of the DNA; this portion of the molecule is sometimes called the sugar-phosphate backbone. The nitrogenous bases extend into the interior, like the steps of a staircase, in pairs; the bases of a pair are bound to each other by hydrogen bonds.
The two strands of the helix run in opposite directions, meaning that the 5′ end of one strand is paired up with the 3′ end of its matching strand. (This is referred to as antiparallel orientation and is important for the copying of DNA.)
Because of the sizes and functional groups of the bases, base pairing is highly specific: A can only pair with T, and G can only pair with C, as shown below. This means that the two strands of a DNA double helix have a very predictable relationship to each other.
When 2 DNA sequences match such that they can stick to each other in an antiparallel fashion and form a helix, they are said to be complementary.

The 5' end has a phosphate group attached to the 5th carbon, while the 3' end has a hydroxyl group attached to the 3rd carbon.

Structures of nucleic acid polymers
Nucleic acids (polynucleotides)
* Polynucleotides are formed by the polymerisation (or adding together) of nucleotide monomers (either deoxyribonucleotides or ribonucleotides) are added together
* A dinucleotide is formed when a condensation reaction occurs between a hydroxyl group (–OH group) at C3 of the pentose sugar on 1 nucleotide and –OH group of the phosphate attached to C5 of the pentose sugar on another nucleotide
* The linkage that connects two nucleotides via a phosphate group is a phosphodiester linkage
* Continual addition of nucleotides produces a polynucleotide strand, with alternating pentose sugars and phosphate groups

Structure of polynucleotides 
A polynucleotide strand has a sugar-phosphate backbone and nitrogenous bases projecting sideways / outwards:
i. Sugar-phosphate backbone
- Made up of alternating pentose sugars and phosphate groups, linked by phosphodiester linkages
- All the pentose sugars and phosphate groups along this sugar-phosphate backbone are identical.
ii. Nitrogenous bases project sideways / outwards from the pentose sugars
- Because the nitrogenous bases at each nucleotide can vary, this gives rise to a specific sequence of nitrogenous bases (A,T,C,G) along the length of the polynucleotide strand; in the case of DNA, it is this unique sequence of nitrogenous bases that carries genetic information for the production of gene product such as proteins, which ultimately influences an organism’s appearance, behavior and development.
A polynucleotide strand has a 5’ end and a 3’ end:
- 5’ end refers to the end with a phosphate group attached to C5 of the pentose sugar.
- 3’ end refers to the other/opposite end with a free hydroxyl group attached to C3 of the pentose sugar.
- During replication or transcription, the genetic information along the polynucleotide strand is read in the 5’ → 3’ direction
- The strong covalent phosphodiester linkages between adjacent nucleotides along the sugar- phosphate backbone confers strength and stability to the polynucleotide strand and prevents it from breaking.
Polynucleotides:
* Polymerisation of nucleotide monomers (also condensation reaction)
* Phosphate group from another nucleotide is attached to C3 on first nucleotide

DNA (Double Helix, Chargaff's Rule)
* Structure: DNA is a double-stranded molecule where two polynucleotide chains are coiled around each other to form a double helix. 
* Sugar: Deoxyribose. 
* Bases: Adenine (A), guanine (G), cytosine (C), and thymine (T). 
* Chargaff's Rule: The amount of adenine (A) is equal to the amount of thymine (T), and the amount of guanine (G) is equal to the amount of cytosine (C). 
* Base Pairing: A pairs with T, and G pairs with C. 
* Double Helix: The two strands are held together by hydrogen bonds between the bases, and the helix is right-handed
RNA (mRNA, tRNA, rRNA)
* Structure: RNA is usually single-stranded, but it can fold into complex 3D shapes. 
* Sugar: Ribose. —> has OH
* Bases: Adenine (A), guanine (G), cytosine (C), and uracil (U). 
* Types:
   * mRNA (messenger RNA): Carries the genetic code from DNA to the ribosomes for protein synthesis. 
   * tRNA (transfer RNA): Transports amino acids to the ribosomes during protein synthesis. (Clover leaf- shaped)
   * rRNA (ribosomal RNA): A component of ribosomes, which are responsible for protein synthesis.

Meselson Stahl Experiments
3 Proposed models of DNA replication
1. Dispersive replication: (Old and new segments dispersed around to form daughter DNA molecules)
* Original parental DNA molecule breaks up into short segments
* Short segments are used as templates for the formation of 2 new daughter DNA molecules
* Segments are then joined together such that the newly synthesised daughter DNA molecules would contain a mixture of old and newly synthesised segments.

2. Conservative replication:  (Original parental DNA molecule is conserved)
* The double-stranded, double helix DNA molecule (original parental DNA molecule) acts as a template for an entirely new double helix DNA molecule to be formed.
* The parent DNA molecule is intact and goes into one of the two daughter cells. The newly formed DNA double helix molecule goes into another daughter cell.

3. Semi-conservative replication: (Both strands of original parental DNA molecule is conserved separately)
* The 2 strands on the DNA double helix molecule separate when the hydrogen bonds between the base pairs are disrupted.
* Each parental DNA strand then acts as a template for the formation of one new DNA strand. Hydrogen bonds form between bases of the original, parental strand and a newly synthesised DNA strand to form the daughter DNA molecule.
* From 1 original parental DNA molecule, it will replicate to form 2 newly synthesised daughter DNA molecules. Each daughter DNA molecule is a hybrid, consisting of one original, parental strand and one newly synthesised, daughter strand.

Messelson and Stahl experiment supports Semi-conservative replication model

Mode of DNA replication: Meselson-Stahl experiment (article) | Khan Academy

DNA Replication process

There are 6 proteins involved in the steps of replication each with specific roles 
* Helicase
* Single-stranded DNA binding proteins
* Enzyme primase
* DNA Polymerase (I and III)
* DNA ligase
Helicase helps with the initial parts of replication . In DNA the specific sites where the origin of replication occurs (ori ) has a specific sequence of nucleotides with a high ratio of it being adenine and thymine .

In this case Helicase will recognise and bind to the ori , where ist breaks the hydrogen bonds between the two strands of complementary bases which form the DNA helix , causing the DNA  molecule to unwind into two separate strands.(Helicase has a complementary binding site to the sequence of dna molecule)

This unwinding will result in a replication bubble with 2 Y shape regions known as replication forks that move apart in opposite directions.
Note: Unwind and unzip are different
* Unzip: involves bond breaking between nitrogenous bases

After the unwinding and unzipping happens Single-stranded DNA binding proteins will attach itself along the two unpaired strands to keep the strands from reforming bonds with one another.
These two separate strands are base templates for the synthesis of 2 new complementary DNA strands with new sequences
These binding proteins will stay until new DNA strands are formed They stay attached to the single DNA strands until DNA polymerase comes along and starts building the new complementary strand.
Enzyme primase will synthesizes a short RNA primer on the DNA template strand during replication. This primer provides a starting point for DNA polymerase to begin addition of the nucleotides.
An RNA primer is a short strand of RNA (usually about 5–10 nucleotides long) that provides a starting point for DNA synthesis. During DNA replication, enzymes like DNA polymerase cannot begin making a new DNA strand on their own and they can only add nucleotides to an existing strand.
* RNA polymerase(unlike DNA polymerase): Can add nucleotides without pre-existing chain during transcription
Specifically the enzymes can only add deoxyribonucleotides to free 3’OH end of a pre-existing polynucleotide chain that has already base-paired with the template because it does not act as a ribosome and cannot start creating its own strands.
DNA polymerase can’t add nucleotides to single-stranded DNA because it needs a free 3’-OH group (hydroxyl group) from an existing nucleotide strand to attach the next nucleotide. The reason why a primer is required because DNA synthesis is a chemical reaction where the 3’-OH of the last nucleotide attaches to the phosphate group of the incoming nucleotide. Without that 3’-OH, the reaction can’t start.
When DNA polymerase moves along the strand and adds new nucleotides, it pushes off the binding proteins because they are no longer needed and the new double-stranded structure is now stable on its own.
Two enzymes will aid in the addition of deoxyribonucleotides and the formation of phosphodiester bonds (A phosphodiester bond is the type of bond that links the nucleotides together in DNA and RNA strands joining one sugar to the phosphate group of complementary bases) between the nucleotides (complementary bases) which are polymerase III and polymerase I .
Extra:Polymerase II is for DNA repair and not involved in replication

Polymerase III is responsible for the addition of the nucleotides , elongating the strand while polymerase I removes primers from DNA strand and replaces it with nucleotides
DNA ligase forms phosphodiester linkages between newly synthesized DNA fragments in the strand to form a continuous strand of DNA.

Leading, lagging & Okazaki
    Leading and Lagging strands

Leading Strand:
* Direction: The leading strand is synthesized in the 5' to 3' direction, towards the replication fault.

* How it's made: Since DNA polymerase can only add nucleotides to the 3' end of an existing strand (the 3' to 5' direction), the strand that is oriented 3' to 5' can be synthesized continuously. The leading strand is synthesized continuously , as the replication fork opens up and DNA polymerase follows it, continuously adding nucleotides in the correct direction.

Lagging Strand:
   * Direction: The lagging strand runs in the 5' to 3' direction (opposite to the direction of the replication fork).

* How it's made: This causes a problem because DNA polymerase can’t add nucleotides in the 5' to 3' direction of the parental strand (since it can only add in the 3' to 5' direction). So, the lagging strand is made in short segments, known as Okazaki fragments. DNA polymerase adds nucleotides to the 3' end of these Okazaki fragments, but once one fragment is finished, the enzyme starts again at a different spot. This creates multiple short segments, which are later connected by an enzyme called DNA ligase to form a continuous strand.
      * Okazaki fragments:
             Okazaki fragments are short segments of DNA that are synthesized on the lagging strand during DNA replication. Since DNA can only be synthesized in the 5' to 3' direction, and the lagging strand runs in the 3' to 5' direction, the synthesis on the lagging strand happens in a bit of a "backward" manner. Instead of being made continuously (like on the leading strand), the lagging strand is made in short, discontinuous segments. These segments are the Okazaki fragments.

Key Differences:
      * Leading Strand: Continuous synthesis in the direction of the replication fork.

* Lagging Strand: Discontinuous synthesis, made in fragments, opposite to the direction of the replication fork.
      *   
Analogy:
-“unzipping” a zipper—one side (the leading strand) just unzips and follows directly. The other side (the lagging strand) needs to start from multiple points along the zipper and work backwards, creating smaller sections that are later stitched together.

Mr Leong’s wisdom can’t be found online: Leading and lagging strands are synthesized in opposite directions as the DNA strands are antiparallel and are opposite directions. Lagging strand has to wait for more of the parental strand to be unzipped for synthesis. Leading strand can be synthesized as parental strand is unzipped.

Typical questions: Why is one discontinuous? Why is one continuous?
Why is the discontinuous strand formed dim fragments?

End Replication Problem
  End Replication Problem:
End Replication Problem is the problem where DNA daughter strands after replication are shorter than the parental strands due to the inability to attach DNA nucleotides after the removal of the RNA primer.

Why it occurs:
Nucleotides can only be attached to the daughter strand at the OH of the 3’ ends
At the extreme 5’ ends of the lagging strand, when the RNA primer is removed by DNA polymerase I, there is no 3’ end available to attach nucleotides to, hence the daughter strand is left shorter than the parental strand. Over multiple replications, the daughter strands will get shorter and shorter, which will eventually result in genetic information to be lost.

How the cell protects itself against this:
         1. Telomeres
         2. Telomerase

Telomeres are a sequence of nucleotides (5’ -TTAGGG- 3’)at the ends of the DNA strands that do not code for anything and are repetitive. It serves as a sort of buffer to delay the erosion of nucleotides which code for important genetic information. (does not solve problem)

Telomerase is an ribonucleoprotein that helps to lengthen telomeres. It consists of:
         * Telomerase RNA which provides a template to guide synthesis of telomere sequences
         * Telomerase reverse transcriptase which catalyses the synthesis of DNA from RNA

Transcript for:Understanding Nucleic Acids and DNA Processes

Transcript for:
Understanding Nucleic Acids and DNA Processes