Lecture 3 - DNA replication Flashcards
What is DNA replication
DNA replication is the complete, faithful copying of the DNA comprising the cell’s chromosomes. It is semi-conservative as discovered in the Meselson and Stahl experiments (each strand of the parental double helix acts as a template). The base on the template strand must be identified and complementary base added.
Describe the process of replication iniation
Chromosome replication starts at specific sites (origins of replication) where the double-helix is opened. DNA is then further unwound to expose single stranded DNA at replication forks where DNA synthesis can then occur. Replication occurs bidirectionally on both strands occurring in a 5’-3’ direction. On one strand the synthesis is continuous and discontinuous on the other.
* The origin of replication is recognized by initiator proteins that open up the double helix and recruits helicases
* DNA helicases unwind the helix to expose single-stranded DNA
* DNA synthesis needs a primer – DNA polymerases can only add nucleotides to an existing 3′ end
The primer is a short RNA strand synthesized by primase
Describe the process of elongation in regards to DNA replication.
- After the RNA primer is synthesized, the sliding clamp is recruited
- DNA polymerase is associated with DNA via the sliding clamp
- Each base in the parental DNA is read by DNA polymerase, and complementary bases are added to the growing strand in a 5′ to 3′ direction
DNA synthesis on the leading strand is continuous but on the lagging strand is discontinuous (Okazaki fragments)
Describe termination in regards to replication.
- Termination of DNA replication occurs when:
○ DNA polymerase encounters DNA that has been replicated
○ Two different forks meet
○ The fork reaches the end of a linear chromosome- Replication complexes are disassembled
- RNA primers are removed and replaced with DNA
DNA ligase connects adjacent strands
How does DNA polymerase catalyse DNA synthesis and how is it directed?
DNA synthesis is catalysed by DNA polymerase
DNA polymerase catalyzes the addition of a new nucleotide to the 3′ OH of the last nucleotide of the growing strand
New DNA synthesis is template-directed – involves recognition of a base in the template strand and addition to the new daughter strand of a nucleotide with the complementary base.
As DNA strands are antiparallel, the template strand has opposite orientation to newly synthesised strand.
Describe the structure of DNA polymerase.
The three domains described as thumb, fingers and palm as together they resemble a right-hand
The palm domain contains the catalytic site for nucleotide addition and forms a cleft in which elongating dsDNA fits
The ssDNA template wraps through the finger domain, which helps position the incoming nucleotide
The thumb domain holds the elongating dsDNA and maintains contact with single strand template necessary for processive synthesis.
What are the main replicative polymerases
The Main Replicative Polymerases
The main replicative polymerase in bacteria is DNA polymerase III - is responsible for synthesis on both strands.
In Eukaryotes there are two main polymerases one for the lagging strand and one for the leading strand:
* DNA polymerase δ - lagging strand synthesis
* DNA polymerase ε - Leading strand synthesis
DNA polymerases are highly conserved and are composed of multiple subunits. All DNA polymerases synthesize only in the 5’-3’ direction.
Describe the mechanism of catalysis by DNA polymerase.
The active site of the DNA polymerase catalyses a phosphoryl transfer reaction. The polymerase links the 5’ phosphate of the incoming nucleotide to the 3’ OH of the growing DNA to form a phosphodiester bond. Nucleophilic attack by 3’ OH on the α phosphate of the incoming dNTP releasing two phosphates as pyrophosphate. Hydrolysis of the released pyrophosphate provides energy for the reaction.
How does DNA polymerase poromote faithful DNA replication?
Specificity of the DNA polymerase active site promotes faithful DNA replication
* Active site of DNA polymerase is selective for correct base pairing
* Correct nucleotide fits precisely in the active site only when base-paired with the template strand
* Mismatches have a different shape to correctly matched bases and don’t fit in the active site as well
* No energy is required for this level of “proofreading.”
Base selectivity ensures an error rate of <1 in 100000
How does exonulease activity proofread?
Proofreading via exonuclease activity
* Replicative DNA polymerases have 3’ to 5’ proofreading exonuclease activity
* DNA polymerisation active site has reduced affinity for 3’OH when incorrect nucleotides present.
* Proofreading exonuclease active site has increased affinity for 3’OH when incorrect nucleotides present.
* Exonuclease active site removes incorrect nt.
* Energy is required to remove the incorrect nucleotide.
DNA synthesis resumed
How are have DNA polymerases evolved?
How does this influence how they are grouped?
DNA polymerases have specialised functions
* DNA polymerase active sites (palm) are highly conserved
* DNA Pols are grouped into families according to the evolutionary lineage of the rest of the protein
* DNA Pols are more similar within groups than within organisms, indicative of early divergence of DNA pol groups
Some have gap repair functions others are involved in primase and repair.
What are DNA helicases?
- Double-stranded DNA is inaccessible to replication machinery
- Unwinding is catalyzed by DNA helicases, which open up the helix and travel along the DNA, continuously unwinding it at the replication fork
- Helicases bind to and move directionally along ssDNA displacing the complementary strand
- DNA helicases - hexameric ring proteins
- Helicase polarity is defined in the direction moved on the strand that is bound
Different polarity suggests independent evolution
What are the differences between DNA helicases in prokaryotes and eukaryotes?
DNA helicases in Prokaryotes vs Eukaryotes
* The E. coli replicative DNA helicase, DnaB helicase, a homo-hexamer, is loaded on to ssDNA by the DnaC helicase loader complex, and moves 5’ to 3’ on the lagging strand template.
* In eukaryotes the core of the replicative helicase is the hetero-hexameric MCM complex (Mini Chromosome Maintenance) is formed of different subunits, MCM2-7 and is loaded onto dsDNA in G1
The full eukaryotic CMG (Cdc45, MCM, GINS) DNA helicase is assembled and activated in S phase, transitioning to encircle ssDNA and moves 3’ to 5’ on leading strand template.
What is the function of Single stranded DNA binding proteins.
- Single stranded DNA exposed by helicase activity can form secondary structures
- Single-stranded binding proteins bind to single-stranded DNA:
○ Single-stranded DNA-Binding protein, SSB, in bacteria
○ Replication protein A, RPA, in eukaryotes - Keeps unwound DNA strands open
- Protects single-strand DNA from nucleases
- Single-stranded binding proteins bind to single-stranded DNA:
What are topoisomerases?
- As DNA is unwound by helicases torsional stress is introduced which results in over winding ahead of the fork – positive supercoiling
- One positive supercoil introduced ahead of the fork for each turn of the DNA helix that is unwound.
- Positive supercoils impede the progress of DNA
Topoisomerases release the overwound DNA by transiently breaking DNA and allowing supercoils to relax
Topoisomerases change the supercoiling status of DNA
* Substrates and products differ only in state of supercoiling
* All types relieve positive supercoiling ahead of fork
DNA gyrase (type II) introduces negative supercoils – and maintains bacterial DNA in a negatively supercoiled state.
