M1 L5: DNA Structure and Replication Flashcards
What properties should the hereditary material have?
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
who’s research contributed to the idea that hereditary material was a component of chromosomes?
Morgan, Sutton/Boveri, Fleming
Who isolated DNA and called it nuclein?
Miescher
Who showed eggs and sperm contribute same number of chromosomes to offspring?
Edmund Beecher Wilson
Why did people think proteins were the hereditary material, not DNA?
20 amino acids vs 4 nucleotides –> proteins should allow for greater complexity
summarize the griffiths experiment. What did it show?
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
summarize the Avery, McCarty, and McCleod experiment. What did it show?
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
Summarize the Hershey-Chase experiment. What did it show?
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
Who helped discover DNA’s structure?
Rosalind Franklin took X ray diffraction images, Watson and Crick proposed double helix
What type of double helix is DNA?
R handed, antiparallel, complementary
What is the result of base stacking? Implications on protein binding?
Base stacking –> major and minor grooves
Larger proteins bind to major groove bc more bases to interact with/more space
what are the purines/pyrimidines and their structres?
Purines: 2 rings, AG
Pyrimidines: 1 ring, CT
What are the 3 forms of DNA and their uses
A form: desiccating (dry conditions) more compact
B form: normal, hydrating conditions
Z form: no real purpose, L handed, assoc w/ TSS
why do mutation rates have to be low but not zero?
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
How do polymerases keep mutation rates low?
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
summarize the Meselson-Stahl experiment. What did it show?
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
What are origins of replication? How are they defined? How many are there per chromosome? Conserved or variable? Why?
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
how many replication bubbles and forks per origin?
1 bubble, 2 forks
summarize the Huberman-Riggs experiment. What did it show?
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
Best characterized origin of rep in bacteria? What consensus seq does it have?
OriC
3 13-mers
4 9-mers
Explain bacterial replication initiation with OriC
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
explain euk. replication initiation with ARS
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
What are consensus seqs
sequences that are highly conserved between distantly related organisms
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?
ARS in yeast (autonomously replicating sequence)
4 - less conserved than in bacteria
Not all predicted origins actually used (20%) –> zones concept
summarize DNA replication
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
What’s an issue with linear chromosomes? How do we solve it?
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
What’s the Hayflick Limit?
cell divisions before apoptosis triggered bc telomeres are too short
How are the ends of chromosomes protected from enzymatic degradation
Seq at end of telomere makes a T-loop –> binds shelterin (protective protein)
Who discovered telomerase? Who first described it?
elizabeth blackburn, carol greider
jack szostak
What types of cells is telomerase most active in?
Germ cells and cancer cells, not somatic cells
How does exercise relate to telomerase? What about cancer cells?
exercise can increase telomerase activity –> longer life
Cancer cells have high telomerase activity –> prevent them from reaching hayflick limit
Differences between PCR and DNA rep in vivo
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
7 components of a pcr reaction?
1) DNA template
2) upstream primer
3) downstream primer
4) buffer
5) water
6) Taq polymerase
7) dNTPs (nucleotides)
2 limitations of PCR?
1) req prior knowledge of sequence (so you can make up/downstream primers)
2) limited to 10-15kb fragments
How can PCR be used for genotyping
use PCR + gel electrophoresis to determine what VNTRs of a gene an individual has (variable number tandem repeats)
describe sanger sequencing. Limitations?
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
describe next generation sequencing. Advantages?
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
