M1 L5: DNA Structure and Replication

Question 1

What properties should the hereditary material have?

Answer 1

1) be in the nucleus, part of chromosomes

2) exists in stable form in cells

3) complex enough to result in organisms

4) able to faithfully replicate itself

5) be mutable –> allows genetic variation

Question 2

who’s research contributed to the idea that hereditary material was a component of chromosomes?

Answer 2

Morgan, Sutton/Boveri, Fleming

Question 3

Who isolated DNA and called it nuclein?

Answer 3

Miescher

Question 4

Who showed eggs and sperm contribute same number of chromosomes to offspring?

Answer 4

Edmund Beecher Wilson

Question 5

Why did people think proteins were the hereditary material, not DNA?

Answer 5

20 amino acids vs 4 nucleotides –> proteins should allow for greater complexity

Question 6

summarize the griffiths experiment. What did it show?

Answer 6

R and S strains of a bacteria (R is nonvirulent, S is virulent) w/ dif strain numbers

mutations can change strain letter but not number

Injected mice with heat treated IIIS and regular IIR –> mice died, IIIS present

Result showed genetic material transferred from IIIS to IIR (transformation). This result cannot be the result of a mutation because they would’ve observed IIS instead of IIIS

Question 7

summarize the Avery, McCarty, and McCleod experiment. What did it show?

Answer 7

Took extracts from the IIIS bacterial cells and destroyed each component in separately –> observed virulence

Sample was virulent in all cases except when DNA was destroyed –> DNA IS TRANSFORMING PRINCIPLE

Question 8

Summarize the Hershey-Chase experiment. What did it show?

Answer 8

Grew bacteriophages with radio labeled phosphorus or sulfur. P–> DNA only, S–> proteins only

Phages attacked bacteria, observed radio labeled P in bacteria, no radio labeled S in bacteria –> DNA IS HEREDITARY MATERIAL

Question 9

Who helped discover DNA’s structure?

Answer 9

Rosalind Franklin took X ray diffraction images, Watson and Crick proposed double helix

Question 10

What type of double helix is DNA?

Answer 10

R handed, antiparallel, complementary

Question 11

What is the result of base stacking? Implications on protein binding?

Answer 11

Base stacking –> major and minor grooves

Larger proteins bind to major groove bc more bases to interact with/more space

Question 12

what are the purines/pyrimidines and their structres?

Answer 12

Purines: 2 rings, AG

Pyrimidines: 1 ring, CT

Question 13

What are the 3 forms of DNA and their uses

Answer 13

A form: desiccating (dry conditions) more compact

B form: normal, hydrating conditions

Z form: no real purpose, L handed, assoc w/ TSS

Question 14

why do mutation rates have to be low but not zero?

Answer 14

low ensures genetic continuity, parents can pass traits onto offspring, and offspring resemble parents

if mutation rate was zero, all organisms would have the same genome, look the same, not be able to adapt to the enviro and have different levels of survival and reproduction –> no evolution by NS

Question 15

How do polymerases keep mutation rates low?

Answer 15

1) high fidelity (usually accurate, rarely make mistakes) / high specificity (specific recognition-addition patterns)

2) DNA proofreading ability: inserting wrong base –> change shape of DNA –> flips messed up area to exonuclease area, remove bases in 3’ to 5’ direction, add correct bases to 3’ end

Question 16

summarize the Meselson-Stahl experiment. What did it show?

Answer 16

Synthesized DNA using entirely heavy nitrogen –> 1 band on cesium chloride density gradient

1 cycle of DNA replication with normal nitrogen –> 1 band on CsCl density gradient for hybrid (half heavy/half light nitrogen) 2 BANDS, heavy and light would show NOT semi conservative

2nd cycle DNA rep with normal nitrogen –> 2 bands on CsCl gradient (some hybrid, some entirely light)

repeat DNA rep with normal nitrogen –> ratio light:heavy N increases

Shows DNA rep is semiconservative

Question 17

What are origins of replication? How are they defined? How many are there per chromosome? Conserved or variable? Why?

Answer 17

Region where double helix separates and replication begins

Defined by seq (AT rich bc only 2 hydrogen bonds)

1/prokaryote chrom, multiple/euk chrom

Highly conserved, mutated seqs can’t be recognized –> DNA can’t be replicated –> organism can’t reproduce

Question 18

how many replication bubbles and forks per origin?

Answer 18

1 bubble, 2 forks

Question 19

summarize the Huberman-Riggs experiment. What did it show?

Answer 19

pulse chase experiment

during DNA replication, pulsed radioactive compound –> incorp into DNA

chase with light version of compound –> incorp into DNA

Pattern of heavy/light incorp was symmetrical w/ respect to origin –> DNA REPLICATION BIDIRECTIONAL

Question 20

Best characterized origin of rep in bacteria? What consensus seq does it have?

Answer 20

OriC

3 13-mers
4 9-mers

Question 21

Explain bacterial replication initiation with OriC

Answer 21

1) DnaA binds to 9-mer region –> change shape and cause stress at 13-mer region –> H-bonds at13-mer region break –> open complex

2) SSB single stranded binding proteins prevent DNA strands from re-annealing

3) DnaB performs helicase activity, brought to open complex by DnaC

Question 22

explain euk. replication initiation with ARS

Answer 22

preRC (pre recognition comples) = 14 proteins, assembles at origin of rep

6 of those proteins form origin of replication complex (ORC)

Cdc6 and Ctd1 bind to ORC, recruit 8 other proteins –> complex separated DNA strands

Question 23

What are consensus seqs

Answer 23

sequences that are highly conserved between distantly related organisms

Question 24

Best characterized origin of rep in euk? How many consensus seq? How are they different from bacteria? Are all origins used? What is this called?

Answer 24

ARS in yeast (autonomously replicating sequence)

4 - less conserved than in bacteria

Not all predicted origins actually used (20%) –> zones concept

Question 25

summarize DNA replication

Answer 25

helicase unwinds DNA –> supercoiling –> topoisomerase (makes organized single stranded breaks in backbone) –> SSB prevents re-annealing

primase adds RNA primer 5’ to 3’

DNA polymerase III synth DNA adding to 3’ OH on RNA primer, add bases to 3’ end of new strand, bound to template by sliding clamp (bc of low processivity), leading/lagging strands

each rep fork has 1 replisome (all proteins req for replication, 1 replisome has 2 DNA pol III - lead/lag)

DNA pol I removes RNA primer/replaces RNA with DNA (both 5’ to 3’) –> gap btwn former-primer and DNA filled with DNA ligase

Question 26

What’s an issue with linear chromosomes? How do we solve it?

Answer 26

Can remove RNA primer from 5’ end of lagging strand, but no 3’ OH group for DNA pol I to replace RNA with DNA –> template strand has 3’ overhang, less and less DNA replicated each time

Solve w/ telomerase: has RNA that complements 3’ overhang –> adds more DNA to leading strand –> room to add primer to lagging strand –> DNA pol III can replicate rest of DNA

Question 27

What’s the Hayflick Limit?

Answer 27

cell divisions before apoptosis triggered bc telomeres are too short

Question 28

How are the ends of chromosomes protected from enzymatic degradation

Answer 28

Seq at end of telomere makes a T-loop –> binds shelterin (protective protein)

Question 29

Who discovered telomerase? Who first described it?

Answer 29

elizabeth blackburn, carol greider

jack szostak

Question 30

What types of cells is telomerase most active in?

Answer 30

Germ cells and cancer cells, not somatic cells

Question 31

How does exercise relate to telomerase? What about cancer cells?

Answer 31

exercise can increase telomerase activity –> longer life

Cancer cells have high telomerase activity –> prevent them from reaching hayflick limit

Question 32

Differences between PCR and DNA rep in vivo

Answer 32

1) PCR uses heat, not helicase and topoisomerase

2) single strands of DNA stabilized by heat, not SSB

3) use pre-formed primers, not primase

4) synthesis by Taq polymerase, not DNA pol III

Question 33

7 components of a pcr reaction?

Answer 33

1) DNA template
2) upstream primer
3) downstream primer
4) buffer
5) water
6) Taq polymerase
7) dNTPs (nucleotides)

Question 34

2 limitations of PCR?

Answer 34

1) req prior knowledge of sequence (so you can make up/downstream primers)

2) limited to 10-15kb fragments

Question 35

How can PCR be used for genotyping

Answer 35

use PCR + gel electrophoresis to determine what VNTRs of a gene an individual has (variable number tandem repeats)

Question 36

describe sanger sequencing. Limitations?

Answer 36

using the same template DNA, set up 4 rxns, each w/ small amt of a dif ddNTP (nucleotide w/o a 3’ OH group –> stops synth once added)

gel electrophoresis –> read bottom to top to get order of bases [5’ to 3’]

low throughput, long time, expensive, requires prior knowledge of seq

Question 37

describe next generation sequencing. Advantages?

Answer 37

Heat template DNA, add capture seqs and primer binding seqs (capture seqs teather DNA to flow cell)

Use one of the templates to synthesize multiple DNA strands on teathered capture seqs —> remove non-teathered strands

add primers, synth new DNA w/ fluorescent nucleotides, excite w/ laser –> identify bases by color

Don’t need prior knowledge of seq (adding your own primers), can read many seq at once