DNA replication Flashcards
Give an overview of DNA replication?
DNA replication is semi-conservative
DNA polymerases use ssDNA as templates to synthesise a complementary strand from correctly paired nucleotides
The OH- on the growing chain nucleophilically attacks the triphosphate and as a result releases pyrophosphate (P2O7 4-)
The chain is extended in the 5’ to 3’ direction
Overview - where does DNA replication occur?
It occurs at replication forks
This is a branch in a replication eye/bubble, that forms when two parental strands separate to allow synthesis of a complementary daughter strand
This replication is bidirectional - the replication forks move apart
Overview - what type of replication model is used in DNA replication?
Semidiscontinuous replication - the parental strands are replicated in different ways
Leading strand - continuously synthesised in it’s 5’ to 3’ direction
Lagging strand - discontinuously synthesised in the 5’ to 3’ direction forming Okazaki fragements (later joined by DNA ligase)
Overview - what is required for initiation of DNA polymerase?
RNA primers - DNA synthesis extends from them
Leading strand needs only one primer
Lagging strand needs multiple primers
Describe prokaryotic DNA?
Double stranded circular DNA
Single replicon - replicated as a single unit
Replicated from a single origin
Bi-directional - goes both ways around the circle to reach the terminus
Describe the different DNA polymerases in prokaryotes?
Pol I, Pol II and Pol III
Structure looks like a hand - when the ‘fingers’ and ‘thumb’ move together it triggers the catalysis
Pol I and Pol II - repair
Pol III - replicase
All add nucleotides in the 5’ -> 3’ direction
All can remove nucleotides in the 3’ -> 5’ direction - using exonuclease
Pol I can remove nucleotides in the 5’ -> 3’ direction - using exonuclease
What does Pol I do in prokaryotic replication?
Pol I has two hydrolytic sites: 3′ - 5′ exonuclease and a 5′ - 3′ exonuclease
3′ - 5′ exonuclease activity allows Pol I to edit its mistakes
Proof reading = high fidelity (accuracy)
5′ - 3′ exonuclease activity allows Pol I to remove RNA primers on the lagging strand and replace with DNA (nick translation)
How does polymerase maintain correctly paired nucleotides?
DNA polymerase senses complementary base pairs via sequence-independent interactions
This occurs in an active site pocket, with a shape complementary to Watson-Crick base pairs
The shallower minor groove allows protein side chains to form H bonds with N atoms of purine bases and O atoms of the pyrimidine bases
If bp were mismatched these interactions would be distorted/disrupted
DNA pol only forms catalytically closed conformation when the correct dNTP binds
What does the DNA polymerase catalytic mechanism involve?
Two metal ions - normally Mg2+ ligands, bound to two Asp side chains in the palm domain
Metal ion A - bridges the α-phosphate of dNTP and the primer’s 3′-OH group
Role: activates primers 3′-OH group for a nucleophilic attack
Metal ion B - is bound to all 3 phosphates of dNTP
Role: orient its bound triphosphate group and electrostatically shield its negative charges
Describe initiation P1 of prokaryotic replication?
DnaA.ATP (initiator protein) binds to 9-mer tandem repeats (AT rich) at the OriC (origin of replication)
This causes strain and denaturation within the 13-mer repeat (forms a melted region)
DnaC (DNA helicase loader) loads 2 hexameric complexes of dnaB (DNA helicase), one to each end of the ‘melted’ region
dnaB helicase ‘unwinds’ the DNA, translocating along the lagging strand, breaking the H bonds between the nitrogenous bases - this uses hydrolysis of NTP for energy (called overhand mechanism)
Describe initiation P2 of prokaryotic replication?
Single-stranded DNA binding protein (SSB) stabilises the ssDNA, preventing reannealing and forming secondary structures (stem loops)
SSB interacts with ssDNA via electrostatic attraction and interacts with the bases via stacking interaction
Primosome moves in the 5’ to 3’ direction, momentarily reversing migration to synthesise short RNA primers (11 nt) - this is needed to initiate each Okazaki fragment
DNA polymerase III holoenzyme can now attach to the primer
What keeps DNA polymerase III holoenzyme in place?
Sliding clamp - it promotes pol III processivity
The β subunit of pol III forms a ring around the DNA that functions as a sliding clamp, preventing pol III from diffusing away
It also greatly increases the rate of nucleotide polymerization
The γ complex = clamp loader
The dimeric ring is a pseudosymmetrical six-pointed star
35 Å diameter hole - doesn’t enter the major/minor grooves = minimises association with DNA
Interior = positively charged
Exterior = negatively charged
Describe elongation of prokaryotic replication?
The strands are synthesised in the replisome (2 pol II enzymes)
Leading strand = continuous replication (5’-3’)
Lagging strand - discontinuous replication (5’-3’)
A primer is continually formed on the lagging strand before each Okazaki fragment - occurs by loop formation
The δ subunit in the clamp loader acts as a ‘wrench’ moving the sliding clamp to the next primer
Prokaryotic replication elongation - how are the RNA primers removed?
RNA primers are replaced by pol I catalysed single-strand nicks, with the nicks sealed by DNA ligase
OR
RNaseH degrades the RNA then pol I removed the primers 3’-ribonucleotide
How does DNA ligase work?
- The adenylyl group of NAD+ is transferred to the amino group of an enzyme Lys residue to form an unusual phosphoamide
- The adenylyl group of this activated enzyme is transferred to the 5′-phosphoryl terminus of the nick to form an adenylylated DNA
AMP is linked to the 5′-nucleotide via a pyrophosphate (not usual phosphodiester bond) - DNA ligase catalyzes the formation of a phosphodiester bond by attack of the 3′-OH on the 5′-phosphoryl group, thereby sealing the nick and releasing AMP
