Gene Expression and Regulation, DNA structure

• DNA synthesis involves the replication of DNA to pass it onto daughter cells.

Pathway for Gene Expression

• The pathway of gene expression involves a series of steps, starting with DNA.
• DNA undergoes replication and transcription.
• Transcription produces pre-mRNA, which is then spliced to form mRNA.
• mRNA is translated into protein.
• Proteins may undergo post-translational modifications to become functional proteins.
• All contribute to gene expression.

Regulation of Gene Expression

• DNA contains genes, which are sections with information.
• DNA converts to RNA through transcription, which includes coding and non-coding RNA.
• RNA converts to proteins through translation.
• Regulation controls gene expression, traits, and cell function.
• Gene expression is regulated through DNA modification, transcriptional regulation, posttranscriptional control, and translational regulation.

DNA Structure

• James Watson and Francis Crick proposed the structural model of DNA.
• DNA is a double-helical structure with two paired strands with complementary nucleotide sequences.
• The double-stranded DNA molecule consists of two spiral nucleic acid chains twisted into a double helix. Twisting gives the DNA its compactness.
• DNA contains millions of nucleotides, molecules with deoxyribose sugar, a phosphate group, and a nucleobase.
• Nucleotides base pair tightly with complementary nucleotides on the opposite strand.
• Adenine (A) pairs with Thymine (T), and Guanine (G) pairs with Cytosine (C).
• One strand is a template for the new strand during replication.
• Nucleotides are linked by phosphodiester bonds, forming a sugar-phosphate backbone.
• A bond forms between the third carbon atom on deoxyribose sugar (3' or three prime) and the fifth carbon atom of another sugar on the next nucleotide (5' or five prime).
• Any part of the sequence can be used to create or recognize its adjacent nucleotide sequence during replication.
• DNA fits within the nucleus by being packed into tight coils called chromatins.
• Chromatins condense into chromosomes during cell division.
• Before DNA replication, chromatins loosen to allow access for the replication machinery to the DNA strands.

The Chemical Structure of DNA

• DNA is a polymer of nucleotide units.
• Nucleotides contain a sugar group, phosphate group, and a base.
• The four bases are Adenine, Thymine, Guanine, and Cytosine.
• Hydrogen bonds hold DNA strands together between bases.
• Adenine (A) always pairs with thymine (T), and guanine (G) always pairs with cytosine (C), but Adenine pairs with Uracil (U) in RNA.
• Bases on a DNA strand act as a code, forming three-letter codons that code for amino acids.
• RNA polymerase transcribes DNA in messenger RNA (mRNA).
• In RNA, Uracil (U) is used instead of thymine (T).
• Protein synthesis occurs in the cytoplasm.
• Translation turns mRNA code into proteins.
• Ribosomes build proteins from coded amino acids via mRNA.

DNA Replication

• DNA replication is a complex process that occurs during cell division (interphase, S phase). Copies are made before the cell divides through mitosis and meiosis.
• DNA replication is a semiconservative process where a parental strand (template) is used to synthesize a new complementary daughter strand. Several protein elements, enzymes, and RNA molecules are used.
• DNA polymerase is the main enzyme for catalyzing the joining of deoxyribonucleoside 5'-triphosphates (dNTPs) to form a growing DNA chain.
• Other proteins are involved for initiation and copying DNA, with capabilities for proofreading.
• DNA replication produces identical DNA helices from one DNA strand.
• DNA replication is essential for cell growth, repair, and reproduction.

The Mechanism of DNA Replication

• DNA replication occurs in three steps:
  • Opening of the double-stranded helical structure and separation of the strands.
  • Priming of the template strands.
  • Assembly of newly formed DNA segments.
• During strand separation, the two strands uncoil at the origin. Several enzymes and proteins prepare the strands for duplication (priming).
• DNA polymerase organizes the assembly of new DNA strands at the end.
• These are general steps that may vary by organism and cell type.
• Enzymes play an important role as they catalyze major stages of the process.
• DNA replication is a vital mechanism for cell function and well-understood in Escherichia coli, similar to eukaryotic cells.
• In E. coli, DNA replication starts at the oriClocus (oriC), where DnaA protein binds while hydrolyzing ATP.

Eukaryotic DNA Replication

• DNA replication is how organisms duplicate DNA to pass to daughter cells.
• Replication ensures that cells receive an exact copy of the parent's genetic material when a cell divides.

Features of Eukaryotic DNA Replication

• Replication is bi-directional and originates at multiple origins of replication (Ori C) in eukaryotes.
• DNA replication uses a semi-conservative method, resulting in double-stranded DNA with one parental strand and a new daughter strand.
• DNA replication occurs only in the S phase and at many chromosomal origins.
• It takes place in the cell nucleus.
• Synthesis occurs only in the 5'to 3' direction.
• DNA strands are manufactured in different directions, producing a leading and lagging strand.
• Lagging strands are small DNA fragments that are eventually joined together called Okazaki fragments.
• Eukaryotic cells have five types of polymerases for replication.

DNA Elongation Complex

• DNA Elongation Complex consists of: PCNA, origin, Pol δ/ε/ΙΙΙ, Pol 1, Nucleotides, Topoisomerase, -SSBS, Helicase, and Okasaki fragments.

The Enzymes of DNA Replication

• Helicases unwind at the start of replication.
• SSB proteins bind to the unwound DNA strands to prevent reformation of the DNA helix.
• Eukaryotic cells contain five DNA polymerases: α, β, γ, δ, and ɛ.
• DNA polymerases α and δ replicate chromosomal DNA, polymerases β and ɛ repair DNA, and polymerase γ replicates mitochondrial DNA.
• DNA polymerase α and δ synthesize the lagging strand via Okazaki fragments.
• The RNA primers are synthesized by polymerase α, which carries a primase subunit.
• DNA polymerase δ synthesizes the leading strand.
• Telomerase, a DNA polymerase with an integral RNA that acts as its primer, replicates DNA at chromosome ends (telomeres).
• DNA topoisomerase I relaxes the DNA helix by creating a nick in one strand.
• DNA topoisomerase II relieves strain by forming supercoils via nicks in both DNA strands.
• DNA ligase forms a 3'-5' phosphodiester bond between adjacent DNA fragments.
• Exonuclease are enzymes that remove nucleotide bases from a DNA chain.

Process of Eukaryotic DNA Replication

• The replication of each linear DNA molecule in a chromosome starts at many origins (one every 30–300 kb of DNA, vary by species and tissue). It proceeds bi-directionally from each origin.
• At each origin, a replication bubble consists of two replication forks that move in opposite directions. The DNA replicated under one origin forms a replicon. Synthesis continues until bubbles merge.
• At the origin, enzymes unwind the double helix making its components accessible for replication.
• Helicase unwinds the helix forming a pair of replication forks.
• The unwound helix is stabilized by SSB proteins and DNA topoisomerases.
• DNA polymerase α, which carries a primase subunit, make required RNA primers.
• DNA polymerase α initiates the lagging strand, by making first the RNA primer and then extending it with a short region of DNA.
• DNA polymerase δ synthesizes the rest of the Okazaki fragment.
• The leading strand is synthesized by DNA polymerase δ.
• The leading strand is synthesized continuously in the 5'to 3' direction. The lagging strand is synthesized discontinuously in the 5'to 3' direction through Okazaki fragments.
• At the completion of synthesis, DNA ligase seals the breaks between Okazaki fragments and around primers to form continuous strands.

DNA Replication Steps/Stages

• Initiation begins with DNA replication.
• Synthesis starts on the template strand at origins.
• Origin sites are targeted by initiator proteins, that recruit additional proteins to form the replication complex around the origin.
• Several origin sites are used for the initiation of DNA replication, forming replication forks.
• The replication complex has the DNA helicase enzyme which unwinds the double helix, exposing the two strands for template replication.
• The DNA primase enzyme synthesizes small RNA primers to start polymerase function.
• The polymerase enzyme grows the new DNA daughter strand.
• Elongation phase where the DNA polymerase grows the new daughter strand. This is done by attaching to the original unzipped template strand and the initiating short RNA primer.
• Able to synthesize a new strand that matches the template, by extending the primer with free nucleotides to the 3' end.
• Reads one of the templates in the 3' to 5' direction, and DNA polymerase synthesizes the new strand in the 5' to 3' direction, known as the leading strand.
• DNA primase synthesizes a short RNA primer along the template strand at the beginning of the template in the 5' to 3' direction, initiating continous synthesis of new nucleotides & extending the new DNA strand.
• Other templates (5' to 3') elongate in an antiparallel direction by adding short RNA primers, which are then filled with other fragments to form the newly formed lagging strand/Okazaki fragments.
• Lagging strand synthesis is discontinuous because newly formed strand is disjointed.
• RNA nucleotides from the short RNA primers must be removed and replaced by DNA nucleotides, joined later by the DNA ligase enzyme.
• During Termination after synthesis the exonuclease removes all RNA primers from the original strand.
• Primers are replaced with the correct nucleotide bases.
• While removing the primers, another exonuclease proofreads the new strands, removing and replacing errors.
• DNA ligase joins the Okazaki fragments to form a unified strand.
• Parent strand ends have telomeres consisting of DNA sequences, which act as protective caps to prevent fusion of nearby chromosomes.
• Telomeres are synthesized by telomerase.
• On completion, parent and complementary strand coil into double helical shape, producing two DNA molecules by passing one strand from the parent molecule and one new strand.

Okazaki Fragments

• DNA strands run in opposite or antiparallel directions.
• At the replication fork, one strand synthesizes in the 5'to3' direction, and the other in the opposite direction, 3'to 5', for continous replication of two new strands.
• However, DNA polymerase can only catalyze the polymerization of the dNTPs only in the 5'to 3'direction.
• The other opposite new strand is synthesized differently
• Discontinuous small pieces of DNA can be joined which are synthesized backward from the direction of replication (Okazaki Fragments).
• Okazaki fragments are joined by DNA ligase, forming an intact new DNA strand (lagging).
• The lagging strand differs from the leading strand primer.
• The Lagging phase is not synthesized by the primer that initiates the synthesis of the leading strand.
• Instead using a short fragment of RNA serves as a primer (RNA primer) for the initiation of replication of the lagging strand.
• RNA primers form during RNA synthesis is initiated de novo plus an enzyme called primase, RNA short fragments with 3-10 nucleotides bases and are complementary to the lagging strand template at the replication fork.
• Okazaki fragments are extensions of RNA primers by DNA polymerase.
• Lagging strand contains RNA-DNA, defining the role of RNA in the replication of DNA.

Replication Fork Formation

• Replication fork is the active DNA synthesis site, where the DNA helix unwinds.
• Multiple origins represent the replication forks.
• Replication fork is formed when the DNA strands unwind by the helicase enzyme, exposing the origin of replication.
• A short RNA primers synthesized by primase and elongation is performed by DNA polymerase
• Replication fork moves in the direction of the new strand synthesis.
• The new DNA strands synthesized in two orientations. 3' to 5' (the leading strands) and 5' (the lagging strands).
• The two sides (leading and lagging) are replicated in two opposite directions.
• The replication fork is bi-directional.
• Leading strand is the continuous synthesis by DNA polymerase.
• It is the simplest strand.
• RNA primer creates by the DNA primase enzyme.
• The primer binds to the 3' start to begin synthesis of the new strand (leading).
• Synthesis is continuous.
• The lagging strand acts has the template synthesis (5' to 3'). It uses RNA primers.
• It includes Okazaki fragments synthesize, the activity of DNA polymerase pieces.
• Strand with the primers.
• Formation of the lagging strand is a discontinuous process the, it the fragmentation of short DNA strands.

DNA Replication Stress

• Reactions of the mechanism and the genome undergoes stress during genome replication.
• Stalled replication and stalled replication fork formation are stress. These events contribute to various stress including:
• Unusual DNA structure.
• Mismatched ribonucleotides.
• Tensions arising from concurrent replication and transcription.
• Inadequate availability of the replication factors.
• Fragile sites of DNA.
• Overexpression/constitutive on ocogenes.
• Unaccessible chromatins.

DNA Proofreading

• Eukaryotes polymerases deal with elongation they have proofreading; (3' to 5' exonuclease activity).
• Erroneous bases detected via 3' to 5' exonuclease removed and correct bases.

Significance of Eukaryotic DNA Replication

• DNA replication is a fundamental genetic process that is essential for cell growth and division.
• DNA replication involve the generation of a new molecule of nucleic acid DNA crucial for life.
• DNA replication is important for properly regulating the growth and division of cells.
• It conserves the entire genome for the next generation.

DNA Replication in Eukaryotes vs Prokaryotes

• Eukaryotic cells posses 25X more DNA than prokaryotic cells
• Eukaryotic cells have multiple origin types and use unidirectional replication, while prokaryotic cells have a single origin type, replicating in the opposite direction simultaneously.
• Eukaryotes have 4+ polymerase types, compared to prokaryotes only using one or two.
• Replication takes up to 400 hours, compared to prokaryote replication speed taking up to 40 mins.
• Eukaryotes have a distinct process for replication the telomeres at the ends of the chromosomes compared to prokarytoes using circular chromosomal DNA, so the have no ends to synthesize.
• Eukaryotic cells only undergo DNA replication during the S-phase, as replication takes place almost continuously.
• Similarities of these processes is, The unwinding of DNA happens before replication for both, DNA polymerase coordinated and the synthesis of new DNA strands, uses the semi-conservative replication pattern, making the leading and lagging strands in different directions/okazaki fragments for lagging strands and they initiation uses a short RNA primer.