MBB 331 Final (old material)

Central dogma

DNA -> RNA -> Protein

Transforming principle

- Griffith's conclusion

- Mouse experiment showed that bacteria transformed avirulent to virulent

- Bacteria can take up foreign DNA from environment, incorporating it and changing traits

One gene one enzyme hypothesis

- Beadle and Tatum that

- Each gene encodes a separate enzyme

- Making nucleus glow, x-ray introduces mutations

Chargaff's Rule

A=T and C=G

Purines

Adenine and Guanine

Pyrimidines

Cytosine, Thymine, Uracil

Bases + sugars in DNA

N-glycosidic bonds

Sugar + phosphates in DNA

Phosphodiester bonds (covalent)

Bp per turn

10.5

Difference between DNA and RNA

RNA has an extra oxygen on the 2 of pentose

How phosphodiester bond is formed

- 5' phosphate binds with free 3' OH of next nucleotide

- gives strand polarity

Chemistry and conditions

pH determines what tautomer form (purines and pyrimidines) they are in

B form of double helix

- Right handed

- 10.5 bp per turn

- Double helix

Major and minor grooves

Due to:

- Antiparallel strands

- Bases attaching to sugar phosphate backbone at an angle

Hairpins

- Secondary structure formed when inverted repeats are present on the same strand

- Unpaired complementary bases and strand folds back on itself

- SS

Denaturation

- Separate strands of DNA with heat

- Nucleotide bonds breaking due to energy being put in

Twisting

- Rotation of bp around helical axis

- Gives DNA double helix shape

Overwound DNA

- More twists

- Positive supercoiling

Underwound DNA

- Less twists

- Negative supercoiling

Compression of DNA

- Shortening distance without breaking covalent bonds

- Bending and looping

- Supercoiling introduced and relieved by topoisomerases

- Protein mediated by packing (histones)

Topoisomerases

Enzymes that allow us to change linking number and superhelicity

Topoisomerase I

- Nicks one strand of DNA

- Thermodynamically favourable because it gets rid of supercoiling

- Changes LK by units of 1

Topoisomerase 2

- Nicks two strands of DNA

- Pass another duplex through break and reseals both strands

- Changes LK by units of 2

- Introduces negative supercoil

Restriction endonucleases

- Enzymes that recognize a specific short DNA sequence and cuts DNA at or near that site

- Generates DNA fragments with either sticky (overhanging) ends or blunt ends

Plasmid or vector

- Small circular double stranded DNA molecule engineered to replicate independently in a host (bacteria)

- Replicates separately from the host chromosome

- Naturally occurring but engineered for gene cloning

Replication origin

- Specific DNA sequence that marks where replication starts

- Allows plasmid to be copied independently of the bacterial chromosome

Plasmid selection markers

- Identification of cells that have successfully taken up the plasmid

- Antibiotic resistant gene

What makes up a plasmid

- Replication origin

- Plasmid selection markers

- Recognition sequences that are targets for restriction endonucleases

Ways to transform bacteria

- Chemical transformation (CaCl2/heat shock) thermal imbalance that drives plasmid DNA into cell

- Electroporation (high voltage electric pulse that creates transient pores in bacterial membrane)

Screenable marker - lac z gene

- Encodes β-galactosidase, which cleaves X-gal to produce a blue color

- If foreign DNA is inserted into the lacZ gene, it disrupts the gene → no enzyme made → colonies stay white

- If no insert is present, lacZ is intact → enzyme made → colonies turn blue

Screenable marker - antibiotic resistance gene

- A plasmid carries a gene that gives resistance to an antibiotic (e.g., ampicillin)

- After transformation, bacteria are grown on antibiotic plates → only cells that took up the plasmid survive, while non-transformed cells die

Gel electrophoresis

- DNA is negatively charged because of the phosphates

- DNA migrates toward the anode in an agarose gel

- Largest pieces of DNA migrate the slowest

Southern blots

Detect DNA

Northern blots

Detect RNA

Polymerase chain reaction

A method of producing thousands of copies of DNA segment using the enzyme DNA polymerase

Genomic DNA

- Entire DNA content of an organism

- Exons, introns and regulatory regions

- Does not reflect gene expression

Complementary DNA

- DNA synthesized from mature DNA

- exons no introns

Sanger sequencing

a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication

Next generation sequencing

entire genomes sequenced using multiple parallel reactions to analyze short segments of DNA and compare the results to known sequences.

Transcriptome

- Complete set of RNA transcripts produced from the genome

Expression vector

- Produces protein

- Gene is transcribed and translated

- Contains regulatory elements that the host cell can recognize and use to make proteins

Promoter/reporter vector

- Measure expression

- Gene activity is monitored not the process itself

Cloning vector

- Store or amplify DNA

- Gene is copied and not expressed

Promoter fusions

- Transcriptional fusions

- Do this when you want to manipulate or change how or where a gene is expressed

- regulatory sequences and coding region

Protein fusions

- Translational fusions

- Do this when you want to investigate how a protein functions or where it is localized

- In frame fusion of coding sequence of 2+ genes

Methylation protecting from endonucleases

Bacteria add methyl groups to specific DNA sequences in their own genome. This modification blocks restriction endonucleases from recognizing or cutting those sites. Foreign DNA (like phage DNA) is unmethylated, so it is recognized and cleaved by the endonucleases.

Homologous recombination

- DNA constructs are designed with regions of homology that match a target site in the genome.

- The cell's recombination machinery recognizes these matching sequences and swaps in the new DNA at that exact location. - This allows precise insertion, deletion, or replacement of genes → creating a transgenic organism.

* Gene knockouts

* Gene Knockins

* Gene Replacements

Double strand break induced editing

- A targeted cut is made in DNA (e.g., using CRISPR/Cas9), creating a double-strand break.

- The cell repairs this break using either nonhomologous end joining (NHEJ) → introduces small insertions/deletions (gene disruption), or homology-directed repair (HDR) if a template is provided → precise gene editing

- Most versatile and widely used

* Loss of function phenotype

* Reporter disruption

* PCR/sequencing

Transposon based insertion

- "jumping DNA" elements that can move from one genomic location to another.

- Efficient but not precise

- A transposase enzyme cuts the transposon out and inserts it into a new DNA site.

- If engineered with a gene of interest, the transposon can randomly integrate that gene into the genome, creating stable insertion mutants or transgenic organisms.

Random integrating (non homologous)

- DNA integrates at random sites via NHEJ

- Unpredictable insertion site

* Antibiotic resistance

* Reporter expression

RNA integration

Sequence specific gene silencing by degrading target mRNA or repressing translation, leading to reduced protein expression

Site directed mutagenesis

Produces a plasmid carrying a precise, predefined mutation that can be propagated and used to study the functional consequences of specific DNA sequence changes

CRISPR

Targeted genome edits through double strand DNA breaks that are required by NHEJ to disrupt genes or by HDR to introduce precise sequence changes, producing stable and heritable mutations.

Affinity purification

Technique to isolate proteins using specific binding partner

Epitope tagging

Adding a short peptide tag to a protein so it can be easily detected or purified using a specific antibody

Western blot

Protein detection

GFP fusion proteins

Protein interest genetically fused to GFP (green fluorescent protein) so the protein can be seen in living cells

Directionality of DNA replication

- Enzyme reads 3'-5'

- New strand forms 5'-3'

Structure of replication fork

- 2 separated template strands

- Leading strand (copied continuously)

- Lagging strand (copied discontinuously) (okazaki fragments)

- Multiple enzymes assemble a replisome

Helicase

Unwinds double helix

Single stranded binding proteins

Stabilize the separated strands

Primase

Synthesizes short RNA primers

DNA ploymerase

Extends DNA 5'-3'

DNA polymerase 1 RNase H

Removes RNA primers and replaces them with DNA pol 1 / RNase H

Ligase

Seals nicks between fragments

DNA pol 3 holoenzyme

10 proteins

- 2 core enzymes with 3 subunits each • One for leading strand, one for lagging strand

- Tau connects the core enzymes

- Beta sliding clamp tethers each core enzyme to a DNA strand

- Gamma complex loads beta sliding clamp onto DNA

Trombone model

The replication of the leading and lagging strands is coordinated by the looping out of the lagging strand to form a structure that acts somewhat as a trombone slide does, growing as the replication fork moves forward. When the polymerase on the lagging strand reaches a region that has been replicated, the sliding clamp is released and a new loop is formed.

Connecting okazaki fragments

On the lagging strand, DNA is synthesized in short pieces called Okazaki fragments. After RNA primers are removed, the gaps are filled with DNA, and DNA ligase seals the remaining nicks by forming phosphodiester bonds, creating one continuous DNA strand.

Control of replication initiation

- Control of DnaA (initiator protein accumulates and starts when there is enough) (ADP inactivates)

- oriC methylation control (Dam/seqA system) (fully methylated initiation allowed but hemimethylated after replication)

- DnaA sequesteration

End replication problem

- During DNA replication, DNA polymerase cannot fully copy the very ends of linear chromosomes because the RNA primer on the lagging strand is removed, leaving no upstream 3' OH to fill in the final gap.

- This causes progressive chromosome shortening each cell division.

Solving end replication problem

- Cells use telomerase, an enzyme that carries its own RNA template.

- It extends the 3' end of the chromosome by adding repetitive DNA sequences (telomeres).

- This provides extra DNA so that important genes are not lost after replication

Termination of DNA replication

- Replication ends when two replication forks meet and DNA synthesis is completed. - In bacteria, termination often occurs at specific termination (Ter) sites bound by proteins that stop helicase movement.

- In eukaryotes, forks simply converge and fuse, and remaining gaps are ligated.

- Finally, chromosomes are decatenated (untangled) by topoisomerases so the DNA molecules can separate properly.

Point mutations

A change in one single nucleotide base in DNA

* Transition

* Tranversion

* Silent

* Missense

* Nonsense

DNA replication with mutation

- DNA pol inserts the wrong base

- If it not repaired before the next round of replication the mutation becomes permanent, one daughter DNA molecule carries the mutation

Frameshift mutation

Indel is not a multiple of 3:

- the reading the frame shifts

- All down stream codons change

- Often creates premature stop codon

In frame indel

Indel is a multiple of 3:

- Reading frame intact

- Effect depends on what region is affected

Large scale mutations

- Large deletions

- Large duplications

- Inversions

- Translocations

- Aneuploidy

What causes large mutations

- Errors during meiosis (non-disjunction, unequal crossing over)

- DNA double strand breaks (radiation, replication repair, faulty repair

- Chromosomal rearrangements

Affect on protein sequence from large mutations

- Gene loss = no protein made

- Gene duplication = too much protein

- Gene disruption = truncated or non functional protein

- Fusion genes = two genes joined together = new abnormal protein with altered function CANCER

Synonymous mutation

*DNA changes but protein same

- Genetic code is redundant (multiple codes for same amino acid)

Non-synonymous mutations

*Nucleotide change that does not change the amino acid sequence

- Missense = one aa replaced with another

- Nonsense = mutation creates a stop codon

Deamination

* Removal of amine (NH2) group from a base

- Base is chemically changed to another

- Spontaneous hydrolysis

- Cytosine to Uracil

- Thymine can't be deaminated due to lack of amino group

Depurination

* Loss of purine from DNA

- Leaves an abasic site

- Water attacks the N-B glycosyl bond between base and ribose

Oxidation

* Reactive oxygen species damage DNA bases

- most common

- ROS arise during irradiation or as byproducts of aerobic metabolism

Alkylation

* Add an alkyl group to either base or phosphate backbone

- N3 of A and O6 of G common sites of alkylation

Radiation

* UV radiation covalently cross links two adjacent pyrimidine (T) in the same strand

- Structural distortion

Base excision repair

DNA repair that first excises modified bases and then replaces the entire nucleotide

nucleotide excision repair

a nuclease cuts out and replaces damaged stretches of DNA

DNA nick (ss break) repair

- Usually repaired accurately using the intact opposite strand as a template

- Fixed by DNA ligase + repair enzymes

- If unrepaired, can stall replication but is generally low risk

Double-strand break repair

- Non-homologous end joining (NHEJ)

- Homology-directed repair (HDR)

* frequently leads to mutations

* cancer is misrepaired

* severe apoptosis

Non-homologous end joining (NHEJ)

- Directly ligates broken ends

- Fast but error-prone → insertions/deletions (mutations)

Homology-directed repair (HDR)

- Uses a homologous DNA template (sister chromatid or donor DNA)

- Accurate repair → can precisely restore or edit DNA

Outcomes of damaged replication fork

- Translesion synthesis (error prone bypass) (replication continues over lesion) TLS pol

- Replication stalls (damage avoidance pathway) (sister chromatid used as temporary template) (homologous like mechanism) (genome preserved)

- Fork collapse (double strand break, repair initiated but not completed ssb causes for collapse leading to dsb) (requires NHEJ or HR) (cancer, genomic instability, chromosomal rearrangements)

- Lesion bypass (replication stalls but picks up again at location downstream)

Programmed generation of dsb during meiosis

- Cells intentionally create dsb to initiate homologous recombination and ensure proper chromosome segregation

- Prophase 1

- Failure = nondisjunction = aneuploidy

Spo2

* Topoisomerase like enzyme

- Creates dsb

- Covalently attaches to 5' DNA after cutting

- No relitigation

transposase

Cuts DNA backbone, leaving single-stranded "sticky ends"

Transposon creates a duplicated target sequence

Because original target DNA was cut in a staggered fashion, filling the gaps duplicates the short sequence on both sides of the inserted tranposon

Genes carried in the transposable element

Determines how it moves, replicates and affects host genome

Structure of transposons

- Insertional sequences (IS)

- Composite transposons

- Site specific recombination

Site specific recombination

- DNA is cut and re-joined at specific short DNA sequences recognized by recombinase enzymes.

- Unlike homologous recombination, it does not require long sequence similarity—just specific "recognition sites."

- The recombinase catalyzes precise insertion, deletion, or inversion of DNA segments.