Central Dogma of Biology and DNA Replication

Central Dogma of Molecular Biology

The process of genetic information flow in cells, summarized as DNA -> RNA -> Protein.

Gene Expression

The process by which genetic information is converted into functional products like proteins or RNA.

Two main steps of gene expression

• Transcription: The DNA sequence is transcribed into messenger RNA (mRNA) within the cell nucleus.

• Translation: The mRNA is transported to the cytoplasm, where it is translated into a protein by ribosomes.

Structure of DNA

Nucleotide polymers, sugar phosphate background, Double helix. Each strand is held together by phosphodiester bonds, and the nitrogenous bases face the “inside” of the helix. Hydrogen bonding interactions between the bases hold the two strands together.

Base Pairing Rule

• Base pairing interaction are between a purine and a pyrimidine

• Purine:

  • Adenine

  • Guanine

• Pyrimidine:

  • Cytosine

  • Thymine

  • Uracil (in RNA)

DNA Replication

• DNA provides directions for it’s own replication

• Each strand can serve as a template for building a new strand during DNA replication

• The two DNA strands in a double helix are antiparallel

• One strand runs in the 5’ to 3’ direction, the other in the 3’ to 5’ direction.

• During replication, each strand results in 2 more strands.

• New nucleotides are added to the 3’ hydroxyl group

• Nucleotides are added to the 3’ end

• DNA polymerase adds the next deoxyribonucleotide monophosphate to the –OH group at the 3’ end of the growing strand and releases pyrophosphate.

• Bonds linking the phosphate groups are broken, releasing energy to drive the reaction.

Models of Replication

Transcription

The process of synthesizing messenger RNA (mRNA) from a DNA template.

DNA in Prokaryotes and Eukaryotes

• Prokaryotes; Single, Circular

• Eukaryotes: 

  • Several, linear

  • Human - 46 chromosomes (~ 6billion base pairs)

  • Eukaryotic chromosomes composed of DNA and numerous proteins (chromatin)

  • Histone Proteins

Organization of Eukaryotic Chromatin Fibers

• Lowest level of organization is the nucleosome

• Nucleosomes are connected to each other by linker DNA

• Nucleosome is folded into ~30nm fiber and these fibers further folded into higher order structures

Mechanisms of DNA Replication

• DNAcontent is duplicated in S-phase of cell cycle

• In humans, takes a few hours for complete replication of DNA

• Takes many enzymes (~12) and is similar for prokaryotes and eukaryotes

DNA Replication; Three Basic Steps

1. Initiation; Unwinding the double helix and separating the two DNA strands

2. Elongation - synthesizing new DNA strands

3. Termination - end of DNA synthesis

DNA Initiation

1. Initiation - Unwinding the double helix and separating the two DNA strands

   1. Occurs at the origin or replication 

      1. Origin is a specific sequence; proteins bind to it + Helix unwinds and opens (replication bubble)

         1. Relieve torsion or stress caused by DNA unwinding

         2. Prevent reformation of double helix

         3. Synthesisize RNA primers (short strand of nucleic acid ~15-40 nucleotides long)

      2. (prokaryotes) Then create pre-replication complex; forming replication bubble with replication forks

         1. Most prokaryotes have a singular circular chromosomes - few million base pairs in size. So ONE origin of replication in prokaryotes.

      3. Eukaryotes typically have multiple (often linear) chromosomes that are much larger in size. MULTIPLE origins of replication in eukaryotes.

DNA Elongation

1. Elongation of new DNA strand is carried out by DNA polymerases

2. Most organisms have multiple DNA polymerase - each with a different role in DNA replication

   1. Replication bubble formation: the replication bubble forms when helicase unqinds and separates the double-stranded DNA molecule at the origin of replication

   2. Primer synthesis by primase: Primase synthesizes short RNA primers on the singel stranded DNA templates, providing a starting point for DNA synthesis by DNA polymerase

   3. DNA synthesis by DNA polymerase: DNA polymerase starts adding complementary nucleotides to the 3’ end of the RNA primer

   4. DNA synthesis by DNA polymerase: DNA polymerase starts adding complementary nucleotides to the 3’ end of the RNA primer, synthesizing the new DNA strand in a 5’ to 3’ direction. As it synthesizes, the replication fork moves along the DNA template.

   5. Leading and lagging strand synthesis: On the leading strand, DNA synthesis occurs continuously in the direction of the replication fork. On the lagging strand, synthesis occurs as short, fragmented segments called Okazaki fragments in the opposite direction of the replication fork. Now, the RNA primers need to be removed + Okazaki fragments need to be linked together.

   6. DNA ligase joins the DNA fragments (forms phosphodiester bonds)

3. In prokaryotes: DNA polymerase III carries out the main DNA synthesis.

DNA Termination

All three steps require coordinated efforts of numerous DNA binding proteins

There are differences between prokaryotes and eukaryotes, but the mechanism is the same.

Write out Prokaryote vs Eukaryote;

#of replication origins, DNA polymerase involved in Elongation, RNA primer removal, Replication Speed, DNA Packaging

Eukaryotic DNA Replication

Prokaryotic DNA Replication

Major Groove

• Wider and more accessible

  • Allows key proteins (helicase and DNA polymerase) in DNA replication to interact with exposed edges of base pairs

Minor Groove

• Less common

• Some proteins involved in DNA replication may interact with the narrower minor groove

Functions involved in initiation of DNA Replication

Function	Protein
Unwind DNA double helix	DNA Helicase
Relieve Torsion/stress caused by DNA unwinding	Topoisomerase
Bind single stranded DNA and prevent reformation of double helix	Single-Strand Binding Proteins
Synthesize RNA primers	Primase

Primase

Type of RNA polymerase enzyme, specifically involved in DNA replication

• Primary function is to synthesize short RNA primers

• RNA primers provide a starting point for DNA polymerase to begin syntehsizying the new DNA strand

• Synthesize RNA using a DNA template

Polymerase

Enzyme that synthesizes nucleic acids

• RNA polymerase – Synthesize RNA

• DNA polymerase – Synthesize DNA

DNA Polymerase

• Always need a template

• Only add nucleotides to the 3’ end of a DNA strand

• Cannot start making a DNA chain from scratch, but require a pre-existing chain or short stretch of nucleotides called a primer

• Proofread, or check their work, removing the vast majority of “wrong” nucleotides that are accidentally added to the chain.

Telomeres

Repetitive non-oding base sequences at the ends of chromosomes

• Allow complete replication of the coding portion of the DNA

  • They are bound by proteins and form looped structures at the end of the chromosomes; protect ends of chromosomes.

• Telomeres shorten with each replication event, and when telomeres become too short to protect the coding section of a chromosome, replication is impeded and cells become dysfunctional

• Telomeres shorten with age

• When telomere reaches limit, cells urdergo senescence or apoptosis

Telomerase

RNA dependednt DNA polymerase that extends telomeres in some cells

• Extends the length of shortened telomeres after DNA replication using TERC

Translation

The process of synthesizing a polypeptide (protein) from mRNA at the ribosome.

Genome

The complete set of genetic information in an organism, cell, or organelle.

Chargaff's Rules

Biochemical rules stating that in DNA, the amount of adenine equals thymine (A = T) and the amount of cytosine equals guanine (C = G).

DNA Replication

The process by which DNA is duplicated, providing each new cell with an identical copy of the DNA.

Semiconservative Replication

A method of DNA replication where each new DNA molecule consists of one old (parental) and one new (daughter) strand.

Leading Strand

The DNA strand that is synthesized continuously in the same direction as the replication fork.

Lagging Strand

The DNA strand that is synthesized discontinuously in fragments (Okazaki fragments) opposite to the direction of the replication fork.