L13-15: Building Blocks of Life Flashcards
What are the functions of DNA?
Genetic code
Storage within cell
Accessibility for transcription
Replication
Meiosis
Genome integrity
How is DNA stored in the cell?
From DNA to histone to nucleosomes + chromatin to further condensation then duplicated chromosome
How does DNA become more accessible for transcription?
Changes binding to histone proteins
What are the 2 main types of variation from meiosis?
Crossing over and chromosome segregation
Which bases are purines and which bases are pyrimidines?
Purines: Adenine, Guanine
Pyrimidines: Cytosine, Uracil and Thymine
What are differences between ribose and deoxyribose?
Ribose has a hydroxyl group on C2
What is a nucleoside composed of?
Base + sugar
Pyrimidine N1 attached to sugar C1
What is a nucleotide composed of?
Base + Sugar + phosphate
Purine N9 attached to sugar C1
Pyrimidine N1 attached to sugar C1
What is the polymeric structure of DNA and RNA?
Linear polymer formed by 3’-5’ phosphodiester bonds
Acidic sugar phosphate backbone
5’-3’ direction
Is DNA or RNA more stable?
DNA >100x
Why is DNA more stable than RNA?
Due to the attraction of the hydroxyl group on the RNA which increases the chance of the chain being broken
What are Chargaff’s rules?
[A]=[T]
[G]=[C]
Number of bases vary between organisms
Who used X-ray crystallography to visualise proteins?
Franklin and WIlkins
Why were X-rays used?
As they have a high frequency and short wavelength
What is the spacing between the inter-plane distance? (Franklin and WIlkins plate 51)
3.4 Angstrom (Å)
How big is the radius? (Franklin & Wilkin plate 51)
10Å
What are the properties of the Watson and Crick model?
2 anti-parallel strands
5’-3’
3’-5’
Right-handed double helix
Backbone outside
Specific base pairing
A-T
G-C
Approximately how many base pairs are there per turn?
10
What are the features of structural stability in DNA?
Hydrophobic effect
Base pair hydrogen bonds
Base pair stacking
Cooperativity
Which part of the DNA is information-rich?
Major groove
How wide is the major groove?
22Å wide
How wide is the minor groove?
12Å wide
What are the 3 types of DNA and what are their properties?
A-DNA: right handed (dehydrated DNA)
B-DNA: right handed
Z-DNA: left handed (lots of salt/supercoiling)
What type of replication occurs in DNA?
Semi-conservative replication
How are mistakes in DNA replication removed?
3’-5’ exonucleases
What is the origin or replication (oriC)
Circular genome
Single origin in E.coli
Five copies of DNA-A binding sequence
AT-rich tandem array of 13mers
DNA-A assembly stimulates unwinding of AT-rich array
How does DNA-B helicase work?
Recruited by DNA-A
Loaded around ssDNA
ATPase-dependent translocation
Strand exclusion model
How does single stranded binding (SSB) protein work?
Loaded on to ssDNA
ssDNA wrapped around SSB tetramers
Prevents secondary structure forming
What is the initiating step of the pre-priming complex?
DNA-A assembly stimulates unwinding of AT-rich array and recruits DNA-B
What is the loading step of the pre-priming complex?
DNA-C is a loading factor that complexes with C-terminus of DNA-B. Following helicase closure around ssDNA, DNA-C hydrolyses ATP and dissociates
What is the activation step of the pre-priming complex?
DNA-G synthesises short strands of RNA known as oligonucleotides during DNA replication
What type of primer is used with the primase enzyme?
RNA
What is primase?
Primase DNA-G, synthsises RNA primer and is recruited by DNA-B
What is toopoisomerase I?
They are enzymes that release tension (relax) supercoiled DNA
How does topoisomerase I work?
It cleaves (breaks) one strand
Passes the cut end under the other strand
Reseals the break
What is topoisomerase II?
Catalyses untangling of the DNA duplexes
How does topoisomerase work?
Cleaves both strands
Passes separate duplex through break
Reseals the break
What are the components of DNA polymerase III? (at alpha unit)
Fingers
Palm
Thumb
5’-3’ DNA synthesis
Error rate 1 in 10^3-10^4
What are the components of DNA polymerase III? (Exonuclease domain)
3’-5’ exonuclease
Proofreading
Reduces error rate 1 in 10^6-10^7
What are the components of DNA polymerase III? (Sliding clamp- beta 2)
35Å diameter hole accommodating dsDNA
Keeps polymerase in contact with DNA
Increases processivity
1-5kb added before falls off
What is the lagging stand problem?
DNA polymerase synthesis 5’-3’ but lagging strand grows 3’-5’
What happens when lagging strand replicates?
It is separated into fragments called Okazaki fragments
How is DNA polymerase III holoenzyme used?
2 DNA polmerases follow DNA-B helicase for coordinated synthesis of leading and lagging strands
What is the Trombone model?
Looping of lagging strand
Lets go after 1000 nts
New loop then formed
Loop lengthens then shortens like a trombone slide
How are Okazaki fragments sealed?
Using DNA ligase and DNA polymerase I (synthesises degrading of DNA/RNA in front)
What are the different DNA damaging agents?
DNA replication stress
Oxygen radicals
Ionising radiation
Chemotherapeutics
Polyaromatic hydrocarbons
UV light
What are the different types of DNA damage?
Base mismatches
ssDNA breaks
DNA adducts (intrastrand crosslinks)
dsDNA breaks (Interstrand crosslinks)
What are the 2 most common cancer genes?
BRAC 1&2 (breast cancer genes)
What is homologous recombination?
Double strand DNA breaks another dsDNA interacts with the end resection so the invasion allows DNA synthesis which repairs the damage, ligation takes place and areas of DNA are crossed over
What is dideoxy sequencing?
2’,3’ dideoxy analogues (ddNTPs)
Form truncated products (chain stops when ddNTPs added)
How did dideoxy sequencing help discover DNA base sequence?
Gel electrophoresis showed bands at the ddNTP areas which allowed for DNA sequencing (used fluorescent dyes)
What are the 3 different types of sequencing methods?
Dideoxy sequencing with fluorescent ddNTPs
Capillary DNA sequencing
Next generation sequencing
