Genetics Unit 2

Why was protein thought to be better suited to serve as

genetic material?

Proteins are made of up to 20 amino acids and DNA is only made of 4 different nucleotides.

What was Griffith’s work with streptococcus pneumoniae?

-Smooth=virulent (fatal) & polysaccharide coat

-Rough=non-virulent

-R strain bacteria transformed into deadly S strain bacteria after taking up genetic material from heat-killed S cells

What was Avery, Macleod, and McCarty’s work?

-transformation only stopped when DNA was destroyed

-argument it could be contaminants

What was Hershey-Chase work?

-bacteriophage with DNA core and protein coat

-radioactive phosphorus-32 in viral DNA entered bacterial cells while radioactive sulfur-35 in viral protein coats remained outside

-bacteria transfer DNA through transduction, transformation, conjugation

Who built double helix model?

Watson and Crick built it after Rosalind Franklin’s lab had a photo

What gives DNA acidity?

-phosphate groups in backbone can release H+ giving it negative charge

What are purines and pyrimidines?

-A and G is purine/double ring

-T and C is pyrimidines/single ring

Explain phosphodiester bonds

-The carbons in the sugar are numbered 1’~5’

- Phosphodiester bond is between 5’ phosphate and 3’ hydroxyl

-give DNA polarity/direction

How do we know DNA is semi-conservative?

-bacteria shifted from 15N heavy to 14N light produces a hybrid in first generation and both hybrid and light (14N 14N) in second generation

-energy for DNA synthesis comes from high-energy phosphate bonds and dNTPs, pol catalyzes phosphodiester bonds

What direction is DNA replication?

-5’ to 3’, nucleotides added by DNA polymerase to 3’ OH

Requirements for DNA to begin replication

-Four dNTPs

 For incorporation into chain

 For energy

-Single-stranded template

- Primer with exposed 3′

hydroxyl

 Adds nucleotides to the 3’ end of

the DNA chain

Explain initiation

1. Initiator protein binds to the origin of replication, short sequence of specific nucleotides

2. DNA Helicase unwinds the helix

3. Replication bubble forms with a replication fork at each end

4. Single-strand binding proteins keep the DNA helix open

5. Primase synthesizes RNA primers

Explain elongation

1. The correct nucleotide sequence is copied from the template strand to a newly synthesized DNA strand

 DNA polymerase III catalyzes phosphodiester bond formation between adjacent nucleotides (polymerization) of new strand

2. Leading strand has continuous synthesis in direction of replication fork (5′-to-3′ direction)

3. Lagging strand has discontinuous synthesis of a 3′-to-5′ strand in small 5′-to-3′ segments

4. DNA polymerase I replaces RNA primer with DNA

sequence

5. DNA ligase covalently joins successive Okazaki fragments

together to complete lagging strand

Unwinding during replication causes supercoiling, explain more

-DNA topoisomerase

relaxes supercoils by

cutting the sugar

phosphate backbone

between nucleotides

-Unwound broken strands

then sealed by ligase

-Synthesis continues

bidirectionally until

replication forks meet

Explain recombination at DNA level

-Independent assortment: each pair of homologous

chromosomes segregates freely from the other

Creates new allele combinations for unlinked

genes

- Crossing over: two homologous chromosomes

exchange portions of DNA

Creates new allele combinations for linked genes

Ensures proper chromosome segregation during

meiosis

Steps of recombination at DNA level

Step 1: Double-strand break formation

 Spo11 breaks phosphodiester bonds of both strands of one

chromatid

 Two non-sister chromatids are shown

Step 2: resection

 5′ ends of each broken strand are degraded by an

exonuclease to create 3′ single-stranded tails

Step 3: Stand Invasion

 Dmc1 helps one single-strand tail invade a non-sister

chromatid and forms stable heteroduplex

 Displacement loop (D-loop) from invaded chromatid is

stabilized by single-strand binding protein

Step 4: Formation of double Holliday junction

 D-loop and other 3′ tail form a second heteroduplex

 DNA synthesis replaces the DNA degraded during formation of the

3′ tails

 DNA ligase reseals the DNA backbones

Step 5: Branch Migration

 Heteroduplex region of both DNA molecules is

lengthened

Step 6: Resolution of the Holliday junction

 Holliday junctions are resolved by resolvase and ligase

What is anticrossover helicase?

disentangles the invading strand from the non-sister chromatid preventing Holliday junction formation

What is heteroduplex?

-region of DNA between breakpoints

 One strand is maternal and other is paternal

 Strands can have mismatches

-two strands of DNA dont break and rejoin at same place

What is gene conversion?

-the physical change of one allele in a heterozygote to the other

 Either allele can be converted

 If heteroduplex region has no gene and parental

alleles are different, gene conversion can occur via

mismatch repair

What is site-specific recombination?

-promotes breakage and rejoining of DNA

molecules at particular sequences

- Single protein called recombinase sufficient to catalyze all breakage

and joining steps

Why is site-specific recombination important?

1. Bacteriophages use site-specific recombination to

integrate their genome into the host bacterium

2. Also important for excision, when DNA is removed from

the chromosome

3. Can create an inversion of a DNA segment

4. If the target site is found at the same position in two

homologous chromosomes, site-specific recombination

can be used to reshuffle regions on non-sister

chromatids

What is transition and transversion?

transition: purine replaced by another purine

transversion: purine replaced by pyrimidine

Forward vs. Reverse Mutation

Forward: wild-type allele to different

Reverse: mutant type back to wild-type (more rare)

(note mutation rate changes based on diff in gene size and gene susceptibility to mutagenic mechanisms)

mutation rate: number of mutations/total sperm

Germ line vs somatic

Germ: reproductive cells, sperm (have more point mutation) and eggs

Somatic: affect individual w mutation

How do we detect mutations?

bacteria as they are small and easy to grow

What do we need to know about replica plating?

-mutations are random and preexisting

-resistant colonies grew in same spot on antibiotic plate, meaning mutation already existed

Prototrophs vs Auxotrophs

-Prototrophs: wild-type bacteria, can grow on minimal media

-Auxotrophs: mutant, need supplement to grow

What is depurination?

-hydrolysis of purine base

What is deanimation?

-remove amine

-change C to U

-normal C-G→ A-T after replication

-base excision repair

What causes thymine dimers to form?

UV light (nucleotide excision), rays also break sugar phosphate backbone of DNA

-note x-rays-double-strandbreaks-homologous recombination or non-homologous end joining

Explain oxidative damage

-8-oxodG mispairs with A

-normal G-C→ mutant T-A after replication

Why do trinucleotide repeats occur?

-slipped mispairing

-excessive amplification of CGG, CAG, CTG, GCC,

GAA are unstable

-the bases are repeated a few or up to 50 times in a

row.

 CGGCGGCGGCGGCGGCGGCGGCGGCGGCGG on one

strand.

-fragile X and Huntingtons

What are mutagens?

-induce mutations

-physical or chemical agent that raises frequency of mutations above spontaneous rate

What are intercalating agents?

-insert between bases

-nucleotide excision

-DNA helix distortion, frameshift inducing lesions

How do mutagens alter DNA?

Base replacement by base analogs (chemical structure almost identical to DNA)

What do hydroxylating agents do?

-add Hydroxyl group

-C now pairs with A

-base excision

What do alkylating agents do?

-add ethyl or methyl group

-modified G pairs with T

-base excision

What is ames test?

-A reverse mutation bacterial assay to assess the mutagenic potential of chemical compounds

-point and frameshift mutations

-high number of reverents (his to his+) suggests mutagen causes mutations

What does base excision repair do?

- removes damaged bases and repairs SSBs

1. An enzyme cleaves an altered nitrogenous base. Another enzyme makes a single nick on the damaged strand.

2. Exonucleases remove nucleotides near the nick

3. DNA polymerase fills in the gap and DNA ligase seals the gap

What does Nucleotide excision repair do?

-detects and removes DNA lesions/distortions

1. A protein complex scans for distortions to double

helix (e.g. thymine dimers) and makes a double nick on the damaged strand

2. DNA polymerase fills in the gap and DNA ligase seals the gap

What does DNA mismatch repair do?

-detects and repairs base mismatches and short ins/del

1. A protein complex detects the mismatch and makes a nick nearby

2. Nucleotides surrounding the mismatch are excised by exonuclease

3. DNA polymerase fills in the gap and DNA ligase seals the gap

What are two repair mechanisms for double-strand breaks?

-Homologous recombination

(S/G2 phases only)

 The same mechanism of meiotic recombination can repair DSBs in mitotic cells using a sister chromatid

-Nonhomologous end-joining (NHEJ) shown here

-Stitches any two broken ends, and sometimes adds or deletes one or a

few base pairs

What happens if our DNA cannot repair mutations?

-breast cancer

-colon cancer

-Xeroderma pigmentosum, avoid sun or skin cancer is guranteed

What do genes specify?

-the identify and order of amino acid in polypeptide chain

-AA chain determine structure and function

What happens if 2 recessive mutations are on the same gene?

-mutant phenotype, fail to complement, otherwise it is wild-type (flies wild-type color is red)

Explain gene function

-supplementation test determines order of genes in biochemical pathway

-pathway halts w missing enzyme

-chemical intermediate that is substrate of this enzyme accumulates

What is missense mutation?

-subs one amino acid for another

-sickle cell disease

What is frameshift mutation?

-insertions or deletions that shift the reading frame, mutations can cancel each other out tho

What are the stop codons and what is nonsense mutation?

-UAG, UGA, UAA

-nonsense is premature stop

Explain the central dogma

-RNA poly transcribes DNA to produce RNA transcript

-Ribosomes translate the mRNA sequence to synthesize a

polypeptide

-translation follows genetic code

Transcription in Prokaryotes- Initiation

-RNA Polymerase binds to the promoter sequence near the beginning of the gene and starts transcription, unwinds double helix

-Promoters are DNA sequences that provide the signal to RNA polymerase for starting transcription

Where does transcription start?

-the +1, moves downstream

-upstream (-) is 5’ and downstream (+ inc in number) is 3’

Elongation

-Nucleotides added 5’ to 3’

-Formation of phosphodiester bonds using ribonucleotide triphosphates (AT P, C T P, G T P, and U T P)

-RNA polymerase moves downstream along the DNA (from the first exon to the last exon)

What are terminators

-RNA sequences that signal the end of transcription

-RNA poly falls off DNA, RNA transcript released

What are enhancers in Eukaryotic genes?

-Can be thousands of base pairs away from the promoter

-Required for efficient transcription

What is primary transcript?

-single strand of RNA that is the result of transcription

In prokaryotes, the primary transcript is

the mRNA

In eukaryotes, the primary transcript is

processed to make an mRNA, 5’ methylated cap and 3’ poly A tail (100-200 bases)

What are introns and exons?

-exons are found in a gene’s DNA and mature mRNA

-introns are sequences found in DNA but NOT in mature mRNA

How does splicing occur?

-splice donor, branch site, splice acceptor

splicing mechanism is 2 sequential cuts remove intron

What catalyzes splicing?

-the splicesome stitches useful parts together

-built from RNA and proteins

-not permanent

Explain translation

-mRNA directs assembly of amino acids into protein

-players are mRNA(messengar), tRNA(adaptor), and ribosomes (rRNA+proteins)

Where does polypeptide/protein synthesis occur

-ribosome, has 2 subunits of RNA and protein

 Small subunit binds to mRNA

 Large subunit has peptidyl transferase

activity, catalyzes formation of peptide

bonds

-Recognize mRNA features that signal

start of translation

- Help facilitate termination by

disassociating from mRNA and

polypeptide

What do tRNAs do?