BIO 251 - DNA

Fredrick Griffith’s Experiment

• 2 strains (S - lethal, R - nonlethal)

• Even when S was heat killed it could still transfer genetic info to R, was lethal

• told us DNA is a transforming substance

Avery et. al. experiment

• DNA vs. Protein

• enzyme analysis of organelles isolated from cell

• process didn’t work when DNase was used but worked when protease was used

• told us DNA is genetic material

Hershey and Chase

• Radioactive tagging

• Phage = DNA and Protein

• blew up cells w bacterial proteins

• when tagging protein: ended up in solution

• when tagging DNA: stayed in pellet → where genetic material is

• Conclusion: DNA is genetic material

Chargaff

• wanted to figure out composition of DNA

• Chargaff’s rules: proportion of A=T and C=G were the same

Bond between base and sugar

N-glycoacidic bond

Bond between sugar and phosphate (one)

phosphoester bond

Bond between phospate from one nucleotide to another

phosphodiester bond

Which are bigger, purines or pyrimadines?

Purines

What is a nucleoside?

base and sugar

What is a nucleotide?

base, sugar, and phosphate

Which part of the nucleotide is at the 5’ end?

Phosphate

Which part of the nucleotide is at the 3’ end?

OH group

From which way to which way do you read DNA?

5’ to 3’

What is DNA held together by?

Hydrophobic bonds → pushes strands together (bases are hydrophobic, phosphates and sugars are hydrophillic)

How are nucleotides connected?

The O in the phosphate group bonds to the C in the sugar and so on

Serretia Lab: What is the ORI

the origin of replication

How many ORI’s in prokaryotes and eukaryotes?

• prokaryotes: one

• eukaryotes: a lotttt

Serretia lab: What is DAP?

It is an enzyme requried to make lysine, which makes protein → e coli needs to grow

What is the bacterial chromosome?

genome for bacteria

What is the PIR gene?

helps the cell replicate, needs gene or else it can’t (on bacterial chromosome)

What is a selection marker?

gene that gives organism a trait

What is KAN? Why can’t serretia grow on it?

It is an antibiotic, serretia cant grow on it unless it has the KAN R. mutation that makes it resistant to KAN

Why can’t serretia replicate plasmid?

The genome doesn’t have the PIR gene

What does the transposase gene do?

It cuts the DNA genes

What does transposase make?

A transposon

From where to where does a transposon go?

It goes from the plasmid to the bacterial chromosome

Where does serretia mutants get their KAN gene from?

E. Coli (already has it), sends KR gene to plasmid of Serretia

What are two ways prokaryotes package DNA?

supercoiling and RNA & Protein scaffolding

What is DNA & Protein scaffolding?

• holds supercoils together

• RNAse → releases scaffold

• Proteinase → releases scaffold

Why does DNA need to be organized?

So it is accessible since it’s so long

What is DNA split into to be organized?

Chromosomes

What is chromatin?

Proteins bind DNA and help tightly package it

What are nucleosomes?

• First level of chromatin

• The basic repeating structural unit of chromatin

• octomer of histone proteins with DNA wrapped around

What is euchromatin?

• Second level of chromatin

• a lightly packed, open form of chromatin that contains active genes and allows easy access for gene transcription

• interphase

• less dense

What is Heterochromatin?

• a tightly packed, dense form of chromatin in cell nuclei that keeps genes turned off and protects chromosome stability

• M - phase

What happens in G1 phase?

• Cell grows

• carries out normal functions

• makes proteins and organelles

What happens in S phase?

• Cell copies its DNA, so each chromosome has two sister chromatids

• DNA replication

What happens in G2 phase?

• Cell continues to grow and prepares for division

• double checks for mistakes

What happens in M phase?

• Cell divides through mitosis and cytokinesis

• produces two daughter cells

• about an hour

What is interphase?

• G1, S, G2

• 6-8 hours

What are the 3 steps of replication in prokaryotes?

Initiation, elongation, and termination

Explain initiation (replication in prokaryotes and eukaryotes)

• DNA replication begins at the origin of replication. Helicase unwinds the DNA and separates the two strands

• Initiator proteins bind to ORI and recruit replication proteins

• Ex: helicase, topoisomerase, and DNA polymerase

Explain Elongation (Replication in prokaryotes)

• new nucleotides added at 3’ end bidirectionally (both strands simultaneously)

• topoisomerase makes nic in DNA

What are the steps of replication in Eukaryotes?

• pre-replication

• initiation

• elongation

• termination

What happens during pre-replication in eukaryotes?

ORC (origin of replication complex → helicase loader (get it clamped on) → helicase

What is helicase?

Separates strands ahead of forks

What happens during elongation in Eukaryotes?

• Primase makes short RNA primers

• DNA polymerase 3 adds new nucleotides to 3’ OH of an existing DNA strand

• uses other strand as template

• incoming nucleotide pairs w/ hydrogen bonding to its complimentary base (contains triphosphate)

What is DNA polymerase?

• Adds nucleotides to DNA strand, builds DNA

• only bonds to double stranded DNA

What is Primase?

• an enzyme that makes short RNA sequences called primers to help start DNA replication

Fill in the blank: DNA polymerase cannot start a new strand from scratch… it must bind to _____ DNA and can only add new nucleotides to an _____ DNA strand at the _____ OH group.

DNA polymerase cannot start a new strand from scratch… it must bind to double-stranded DNA and can only add new nucleotides to an existing nucleic acid DNA strand at the 3’ OH group.

DNA synthesis is _____ for leading strands

continuous

DNA synthesis is _____ for lagging strands. AKA what?

• discontinuous

• okazaki fragments

What does nuclease do?

It removes the primer

What does DNA polymerase 1 do?

fills in gaps

What does ligase do? What does it couple up with to work?

• Ligase connects the sugar-phosphate backbone

• Couples with ATP hydrolysis for energy; the reactions couple together to work

What does the sliding clamp do?

It keeps DNA polymerase on track

What do single-stranded DNA-binding proteins do?

They keep lagging strand linear until DNA polymerase comes along

What does topoisomerase do?

untwists ahead of replication

Wrong nucleotides won’t what?

they won’t create an H-bond

How many domains does DNA polymerase have? What are they?

2 domains

1) polymerase

2) exonucleus

What happens in the polymerase domain of DNA polymerase?

• Works 5′ → 3′ and adds nucleotides to the growing DNA strand.

• faster domain

What happens in the exonucleus domain of DNA polymerase? (proofreading)

• Works 3′ → 5′ and removes & replaces incorrect nucleotides for proofreading.

• slower domain

What are telomeres?

they are repeating short sequences at the end of eukaryote chromosomes

What does telomerase do? What are its components?

• adds DNA repeats to the ends of chromosomes → extends telomere

• DNA and protein

How does telomerase use protein and DNA?

the RNA component helps telomerase bind to telomere end and serves as a template for the protein to add new DNA nucleotides to the end

What are “putative mutants”?

potential mutants

What are the three types of DNA repair?

• Mismatch repair

• Excision repair

• Homologous recombination repair

When is mismatch repair? What are the outcomes?

• occurs simultaneously with replication

• Outcomes: no repair, repair of only one strand, or repair and excision of only the newly synthesized strand

How do prokaryotic cells know which nucleic acids are correct in DNA?

• older DNA strand is methylated

• new strand is not yet methylated

How do eukaryotic cells know which nucleis acids are correct in DNA?

• they scan upstream for gaps where different ORI’s meet, or nicks in newly made strands

What is excision repair? What are the two types?

• repairs DNA lesions/damage that occurs later

• spontaneous hydrolysis reactions

• UV or Xrays

What are 2 types of spontaneous hydrolysis reactions (excision repair)

• Depurination

• Deamination

What is depurination?

• When a purine base (A or G) is removed from DNA, leaving behind an empty spot in the DNA strand

• bond between the sugar and purine base breaks

What is deamination?

• Removal of an amino group (–NH₂) from a DNA base

• changes the base into a different base

Explain UV or X-ray excision repair

• provides energy for bond rearrangements

• if damaged → forms thymine dimer

What is a thymine dimer?

chemical bonds between two neighboring thymine bases in a DNA strand

What enzymes take part in repair?

• DNA endonuclease (excision)

• Helicase (excision)

• DNA polymerase (resynthesis)

• ligase (ligation)

What is DNA endonuclease?

• An enzyme that cuts DNA within the strand, breaking the bonds between nucleotides

• removes damaged or incorrect sections

What is homologous recombination? Where does it happen?

• A DNA repair process where a damaged DNA strand uses a similar or matching DNA sequence as a template to repair itself.

• similar to crossing over in meiosis

• Happens in naturally-competent bacteria

What are the bonds between C-N called?

peptide bonds

Phosphoanahydride bonds

bonds between phosphate groups

Which bases are purines?

Adenine and Guanine

Which bases are Pyrimadines?

Thymine, Uracil, and Cytosine

Where is the N-terminus?

The N-terminus is the starting end of a protein chain with a free amine group (-NH₂)

Where is the C-terminus?

the C-terminus is the final end with a free carboxyl group (-COOH)

How many hydrogen bonds are between Adenosine and Thymine?

2 H-Bonds

How many hydrogen bonds between cytosine and guanine?

3 H-bonds

What are essential components of DNA structure?

• DNA is made of nucleotides

• Each nucleotide contains a deoxyribose sugar, phosphate group, and nitrogenous base

• DNA forms a double helix

What is at the 3’ end and 5’ end?

• 3’ → -OH group

• 5’ → Phosphate group

What roles do Hydrogen bonds play in DNA structure and function?

• Structure: holds strands together

• Function: They are weak enough to allow the strands to separate during replication

What general mechanism for DNA replication is suggested from this model of DNA?

• The double helix can unzip, with each original strand serving as a template for a new complementary strand.

• This leads to semiconservative replication (one original parent and one new strand)

How is the packaging problem solved in eukaryotes?

• DNA wraps around histone proteins to form nucleosomes.

• Nucleosomes fold and organize into increasingly compact forms of chromatin.

Components of a nucleosome:

About 147 base pairs of DNA wrapped around a core of 8 histone proteins (a histone octamer).

What did the Meselson and Stahl experiment discover?

• They grew E. coli in heavy nitrogen (¹⁵N) and then moved them to light nitrogen (¹⁴N).

• They separated DNA by density after each generation.

• Results: After one generation, DNA was intermediate density (¹⁵N/¹⁴N). After two generations, there was intermediate and light DNA.

• This supported the semiconservative model: each new DNA molecule has one old strand + one new strand.

What is included in termination in eukaryotes?

Ligase seals gaps between DNA fragments.