WEEK 6 Flashcards
(35 cards)
List the replication fork proteins
- DNA polymerase ɛ, α and δ
- Gyrase
- DNA helicase
- Single stranded DNA-binding protein
- RNA primer
- sliding clamp
- Clamp loader
DNA polymerase delta and epsilon function
Template DNA strand synthesis (from lagging to leading strand)
DNA polymerase alpha function
Initiates nuclear DNA synthesis and DNA repair
DNA gyrase function
Helps prevent DNA over-twisting and relieves torsional stress
Sliding-clamp protein
Allows stable binding of DNA polymerase to the DNA
Clamp-loading proteins
AIDS in attaching sliding-clamp proteins
Sliding Clamp protein function
Allows stable binding of DNA polymerase and hence efficient strand synthesis
More tightly wound DNA (dark bands)
Heterochromatin
Less tightly wound DNA, more accessible DNA (light bands)
Euchromatin
Ends of chromosomes
- called telomeres
- have tandemly repeated G-rich sequences (TTAGGG)
Centromeres
- separate the p and q arm (p is smaller, q is longer)
For a chromosome top be copied it requires: (3)
- centromere
- replication origins - initiation
- telomeres (telos = end)
Initiation of Eukaryotic DNA replication
- assembly of the replication proteins is ordered and begins at precise chromosomal locations (origins)
- length and sequences vary greatly between species
- origins are AT-rich
- as there are many origins, replication requires coordination
- Origin recognition complex = multi-protein complex, bonds origin and separates DNA strands by recruiting replication proteins
- in eukaryotes, DNA replication proceeds in both direction with multiple points of origin
- to initiate replication DNA is licensed at origins (can be licensed only once
- DNA helicase recruited to DNA by S phase specific proteins to ensure DNA replication occurs only in S phase once (cannot occur again)
As replication fork forms in pairs, what is created?
Replication bubbles
What is telomerase
- specialised enzyme with a a catalytic subunit (TElomerase Reverse Transcriptase - TERT)
- along with an RNA component, completes chromosome end to maintain chromosome length
CAF - 1
- chromatin assembly factor 1
- brings histones to replication fork, assemble to form nucleosomes to chromosomes
Model for formation of DNA Polymers (6)
- Elongation occurs by addition to 3’ end (free 3’ -OH)
- Enzyme = DNA polymerase
- Substrate = deoxynucleoside triphosphate (dNTP)
- Requires a “template” strand to base pair
- Requires short double stranded NA (primer)
- Requires Mg2+
Lack of 3’ OH group leads to …
… termination of DNA synthesis
PCR components and function
- dsDNA template = DNA you want to copy
- primers to provide 3’OH (needs two sets, one for each DNA strand)
- enzyme (Taq DNA polymerase) to make DNA
- dNTPs - substrates (dATP, dCTP, dGTP, dTTP)
- Mg2+ for enzyme activity
- Buffer to maintain pH
- thermonuclear to heat and cool
Coding strand
5’ to 3’ (also called sense)
Why Taq Polymerase
Stable protein activity up to 95°C and high processivity
PCR 3 stages
- Denaturation of DNA ~95°C (expose templates)
- Primer hybridisation (binding/ annealing) ~50°C
- DNA synthesis (extension) ~72°C
RNA structure (components) and features (8)
- uracil replaces thymine found in DNA
- sugar is ribose
- single-stranded
- phosphate: -ve
- bases flat
- relatively hydrophobic
- forms H-bonds
- absorb at 260nm
From what direction is RNA read
5’ to 3’
