Lecture Notes: DNA, Transcription

Introduction by Catherine Skelding

Catherine Skelding is a cancer cell biologist and biochemist.
She expresses her love for teaching HUBs 226 and her fascination with molecules and their functions within cells.
The course will cover DNA, protein, and enzymes.

Teaching Style and Lecture Structure

Varied educational styles: words, pictures, animations, and videos.
Lectures are divided into chunks with activities every 10-15 minutes.
Activities include example exam questions to consolidate learning and understand exam question design.
Revision points and activities: flow charts, tables, matching exercises.
Questions are welcome via email.
Revision lectures offered for lectures 8-16, with example exam questions designed based on emailed questions.
Practice exam questions will be available as quizzes.

DNA Replication: Replicating Genetic Material and DNA Repair

Some content will be a recap from earlier courses with more detail.
Focus on why inhibiting certain molecules affects diseases and can be used for treatments.
Learning goals include understanding the role and structure of the nucleus, DNA replication, drugs targeting DNA replication, telomeres and telomerase, and main DNA repair pathways including basic excision repair, nucleotide excision repair, homologous recombination, non homologous end joining and mismatch repair.

Recap of the Nucleus

The nucleus contains DNA and is bounded by a nuclear envelope consisting of an outer and inner nuclear membrane.
The nuclear envelope is connected to the endoplasmic reticulum.
A green mesh beneath the inner nuclear membrane is the site of DNA replication.

DNA Structure

DNA is a double helix wound around histone proteins, forming chromatin and chromosomes.
DNA is made up of two long polynucleotide chains composed of four types of nucleotides with a sugar-phosphate called deoxyribose and a nitrogen-containing base.
These include adenine (A), thymine (T), guanine (G), and cytosine (C).
The strands have a five prime and a three prime end and are orientated anti parallel to each other, held together by hydrogen bonds.
Adenine always binds to thymine, guanine to cytosine.
This structure allows for easy duplication and is the mechanism for heredity.
Replication is semi-conservative, with each daughter cell inheriting a new DNA double helix containing one original and one new strand.

DNA Replication Process

DNA synthesis begins at replication origins (or replication bubbles) and proceeds bidirectionally.
The replication fork is the localized region of replication that moves along the parental DNA.
Initiator proteins start DNA replication and recruit DNA helicase.
DNA helicase unwinds the DNA helix at adenine-thymine rich regions.
Single-stranded DNA binding proteins bind to single strands, preventing hairpins and keeping bases exposed.
DNA polymerase synthesizes DNA in the five prime to three prime direction, adding complementary deoxyribonucleotide triphosphates.
Complementary base pairing: A to T, G to C.
Most DNA polymerases proofread with a three prime to five prime exonuclease activity.

Leading and Lagging Strands

The leading strand is synthesized continuously in the five prime to three prime direction.
DNA primase lays down a primer, and DNA polymerase three synthesizes the DNA.
The lagging strand is synthesized discontinuously because no three prime to five prime DNA polymerase has ever been identified.
DNA primase lays down a primer, and DNA polymerase three synthesizes short Okazaki fragments.
An exonuclease removes the primer, and DNA polymerase one fills in the gaps.
DNA ligase joins the fragments together.

DNA Topoisomerase

DNA topoisomerase introduces swivel point by breaking phosphodiester bond in DNA strand.
This lets the DNA rotate without supercoiling, which would produce that torsional stress and fragment replication.

Flow Chart of DNA Replication Steps

Initiator proteins initiate DNA replication.
DNA helicase separates the strands.
Single-stranded binding proteins keep strands separated.
DNA polymerase replicates DNA.
DNA topoisomerase prevents supercoiling.
Leading strand: DNA primase lays down RNA primer, DNA polymerase three adds nucleotides continuously.
Lagging strand: DNA primase creates RNA primer, DNA polymerase three synthesizes Okazaki fragments, exonuclease removes primer, DNA polymerase one fills gaps, DNA ligase joins fragments.

Drugs That Target DNA Replication

Cytosine arabinoside (cytarabine, AraC) inhibits DNA polymerase and is used to treat leukemias.
Fluoroquinolones (e.g., ciprofloxacin) inhibit bacterial topoisomerases, fragmenting bacterial DNA.

Telomeres and Telomerase

Telomeres are repeated DNA sequences at the end of chromosomes that prevent loss of coding information during replication.
Telomerase is an enzyme in germ cells, stem cells, and some white blood cells that adds repeating sequences to the three prime end of DNA.
Cancer cells may have active telomerase, contributing to their replicative immortality.

DNA Damage and Repair

DNA can be damaged by smoking, ionizing radiation, ultraviolet light, chemicals, and errors in replication, transcription, and translation.
The cell has multiple pathways to repair alterations due to the DNA double helix containing two copies of genetic information.
Main types of DNA repair: base excision repair (BER), nucleotide excision repair (NER), non-homologous end joining (NHEJ), homologous recombination (HR), and mismatch repair (MMR).

Base Excision Repair (BER)

Repairs specific damaged bases, such as deamination of a base pair.
DNA glycosylase travels along DNA, flips out bases and recognizes damaged base.
It then recruits enzymes which will cut either side of the damaged nucleotide, DNA helicase unwinds, DNA polymerase puts in the correct base pair and DNA ligase synthesizes.

Nucleotide Excision Repair (NER)

Repairs large, bulky, helix-distorting lesions, such as pyrimidine dimers from ultraviolet light damage.
Sensed by multi-protein complex or when RNA polymerase stalls during transcription.
Enzymes cleave the strand on either end of the lesion.
DNA helicase unwinds the strands, damaged region gets removed, DNA polymerase synthesizes the gap and DNA ligase joins the fragments together.

Double-Stranded Breaks

Double-stranded breaks are repaired by non-homologous end joining (NHEJ) or homologous recombination (HR).
NHEJ: broken ends are recognized by Q heterodimers which brings them together.
Ends are processed, nucleotides lost, and joined covalently.
Occurs any phase of the cell cycle but is error prone.
HR: high-fidelity pathway that uses the homologous chromosome as a template.
Can only take place during S and early G2 phases when the homologous chromosome is near the broken strand.
Strand exchange/invasion where broken strand joins to homologous chromosome, DNA polymerase synthesizing DNA, and DNA ligase joining strands.
BRCA proteins involved in this pathway.

Mismatch Repair (MMR)

Removes replication errors missed by the replication machinery, where non-complementary base pairs are involved.
MutS recognizes the mismatch and binds to the mismatch base pair.
MuT S scans DNA for nicks, triggers degradation back to the mismatch.
DNA polymerase fills the gap and DNA ligase joins the fragments.

DNA Repair Pathways Summary

Base excision repair (BER): DNA glycosylase recognizes damage, endonuclease excises the damaged base, DNA polymerase fills the correct nucleotide and DNA ligase joins it together.
Nucleotide excision repair (NER): A large bulky helix distorting lesion is sensed, enzymes excise bases, DNA helicase unwinds helix, DNA polymerase synthesizes and DNA ligase joins.
Non-homologous end joining (NHEJ): Q heterodimer binds recognized and brings together broken ends, losses of nucleotides and covalently joining.
Homologous recombination (HR): Strand exchange where undamaged DNA used by DNA polymerase to synthesis and the strand is released and the strands are joined by DNA ligase.
Mismatch repair (MMR): Mismatch base, GANS DNA and Mutess and Mutel degrades DNA back to where degradation can occur by and DNA polymerase and ligase.

Transcription: Moving from DNA to Proteins

Central dogma of molecular biology: DNA codes for protein via transcription and translation.
Ribonucleic acid (RNA) is a single linear polymer composed of four types of nucleotides containing sugar phosphate called ribose and four nitrogen-containing bases including cytosine, adenine, and guanine.
Uracil (U) replaces thymine.
RNA folds into complex 3D shapes.
Types of RNA: messenger RNAs, ribosomal RNAs, transfer RNAs, small nuclear RNAs, small nucleolar RNAs, microRNAs, small interfering RNAs, PWE interacting RNAs, and long non coding RNAs.
Focus on messenger RNAs, transfer RNAs and small nuclear RNAs.

Transcription Process

Transcription produces messenger RNA complementary to one strand of DNA, carried out by RNA polymerase.
Transcription begins with opening and unwinding of portion of the DNA double helix called transcription bubble where transcription occurs using one strand as a template.
Enzyme RNA polymerase is used to carry out the transcription by catalyzing the phosphodiester bonds and the help from transcription factors.

Complementary Base Pairing

A is complementary to U.
G is complementary to C.
T is complementary to A.
C is complementary to G.

Transcription Initiation

RNA polymerase requires transcription factors to initiate transcription.
The promoter that contains the TATA box is the starting and binding point for RNA and polymerase that is 25 nucleotides downstream downstream.
TF2D binds at TATA box.
TF2B, H and E help recruit and align RNA polymerase.
TF2H unwinds helix and exposes nucleotides.
TF2H phosphorylates RNA polymerase to cause factors to be released and transcription will begin.
We also have chromatin remodeling complexes and histone modifying enzymes.
Acticator proteins are locate nucleotides and the medium protein aligns to the complex.

RNA Processing

Transcription elongation requires accessory proteins to stabilize RNA polymerase and drive transcription forward.
Messenger RNA processing: RNA splicing removes introns, five prime end capping, polyadenylation at the three prime end.
Addition of poly A tail takes place by an enzyme called poly A polymerase.

RNA Splicing

A machinery called the spliceosome is used to perform RNA splicing.
The spliceosome recognizes splice site at edges and cuts phosphate backbone triggering a loop or lariat where the spliceosome holds the fragment and releases intron while the exons come together.

Alternative Splicing

Alternative splicing increases coding potential without more genes.
The primary transcript can be spliced in different ways to produce variant messenger RNAs for increased protein functions within tissues.

Transcriptional Regulators

Transcriptional regulators regulate transcription and recognize specific DNA sequences.
Messenger RNA is relatively unstable, allowing rapid change due to stimuli such external changed conditions.
Repressors and activators are used.

Molecular Processes Summary

The enzyme that drives transcription is RNA polymerase two.
Transcriptional activators bind hundredds/thousands of base pairs upstream of where transcription starts and is part of the initiation complex.
Transcription factors, TF2H, TF2B and D bind at the TATA box.
The promoter is the region that helps position RNA polymerase 25 nucleotides downstream of promoter.
TF2H is the factor involves unwinding helix that lets other molecules to bind.
Activator protein binds to mediator to help align complexes at correct spot.
The binding of activator causes TF2E to activate TF2H where the phosphorylation of RNA polymerase cause transcription to elongate.
The seven mthylguanosine is then added.
When the terminator sequence is achieved, you can then perform poly a polymerase adds to messenger with cap and tail.
The splicing then occurs by thespliceosomes that slices introns and joining. And that is then achieved to have a fully mature messenger RNA.