Lecture 5 Flashcards
What does DNA polymerase require?
Substrate - dNTPs
Template - parental DNA template strand to direct the correct nucleotide sequence
What is DNA polymerase?
An enzyme that catalyses the addition of 1 nucleotide at a time to the 3’ end of a DNA chain
What is a primer?
RNA primer - short chain of ribonucleotides
- provides the free 3’ -OH end in which deoxynucleotides are added to
What kind of bond is catalysed by DNA polymerase?
Phosphodiester bond
What are template strands read as and what are new strands read as?
Template strands are read 3’-5’
New strands are read 5’-3’
How many DNA polymerases does E.coli contain and what are their functions?
3
DNA polymerase I - repairs DNA and participates in DNA synthesis of one strand
DNA polymerase II - DNA repair
DNA polymerase III - Major DNA replication enzy,e
- responsible for strand elongation
- exhibits 3’-5’ exon lease activity which allows proof reading
Where is DNA polymerase bound to until DNA synthesis is complete?
The replication fork
What is the name given to the entire replication machinery?
Replisome
What accessory proteins are involved in replication?
Primase - provides 3’ -OH end
Helicase - unwinds double stranding of DNA
Single-strand binding protein (SSB) - binds to single stranded DNA and prevents it from reforming the double helix
DNA ligase - joins nicks in a DNA strand (ligation)
What are the three steps in DNA replication?
Initiation
Elongation
Termination
What happens during initiation?
Assemble of proteins at the start site
Opening of DNA at the origin
Helicase uses energy from ATP to unwind double strands of DNA
SSB protein then binds to the single stranded DNA preventing renaturation
DNA primase attaches to DNA and synthesises a short RNA primer
What happens during elongation?
Replisome moves along the DNA simultaneously unwinding and synthesising new strands
Both strands are elongated by DNA polymerase III which adds nucleotides to the growing strands - the enzyme is a dimer - each half synthesising a strand
The primers are removed and filled in by DNA polymerase I
The fragments are joined by DNA ligase
What happens during termination?
Disassembly of proteins and growing daughter strand
The two replication forks meet approximately 180* opposite the replication origin
There are several terminator sites which stop the movement of the forks by binding to an inhibitor of helicase
Once replication is completed, the two daughter DNA circles remain interlinked (catenated)
They are unlinked by a type II DNA topoisomerase
How does DNA replication differ in eukaryotes?
Mechanism is similar but replication is slower - DNA is packaged
- Okazaki fragments are shorter, 100-200 nucleotides
Replication only occurs in the S-phase of the cell cycle
Several origins of replication start simultaneously
- origins have a simple consensus sequence which binds the replication complex
5 DNA polymerases
Accessory proteins that function like some of the prokaryotic proteins
What are the 5 eukaryotic DNA polymerases?
Alpha - synthesis of lagging strand, contains subunit with primase activity Beta - DNA repair Gamma - mitochondrial DNA synthesis Delta - synthesis of leading strand Epsilon - DNA repair
