8. DNA Replication Flashcards
What are the requirements for DNA replication?
Single-stranded template Deoxyribonucleotide triphosphates Mg++ Replisome (protein complex) Primer with free 3' end hydroxyl group
What sequence is indicative of an origin of replication?
Short AT rich region
What is the function of DNA helicase?
Separate the DNA strands
Uses ATP
What is the function of single-strand binding proteins?
Prevent the strands from re-associating
What is the function of topoisomerase?
Regulates twisting of DNA
Can cut DNA
What is the function of primase (RNA polymerase enzyme)?
Makes a primer (RNA starter strand)
After replication, RNA primer is removed and the gap is filled by DNA polymerase
What is the function of DNA polymerase?
Uses single strand DNA as a template
Reads template 3’ to 5’
Makes new DNA from 5’ to 3’
Aligns and adds nucleotides along template which specifies the sequence of the new chain
Catalyses formation of phosphodiester bonds
What is the role of PCNA?
Acts as a sliding stamp to keep DNA Polymerase closely associated to the template
How are errors prevented?
Substrate specificity: Catalysis only occurs when correct dNTP is bound
‘Proof reading’: 3’ to 5’ exonuclease activity
Describe lagging strand synthesis
Primase makes a new primer at regular intervals
DNA polymerase replicates the template from the primer producing a new strand in the 5’ to 3’ direction
DNA Polymerase is blocked by the proximity to the next primer
Primers removed, gaps filled and backbone joined
What is the role of Polymerase alpha?
Initiating replication
Associates with primase
Replicates DNA by extending primer 5’ to 3’
What is the role of polymerase epsilon?
Replicates leading strand DNA by extending primer 5’ to 3’
3’ to 5’ exonuclease activity
What is the role of polymerase delta?
Replicates lagging strand DNA by extending primer 5’ to 3’
3’ to 5’ exonuclease activity
What is the role of polymerase beta?
Involved in DNA repair
What is the role of polymerase gamma?
Replicates mitochondrial DNA
What is the role of Rnase H1?
Removes most of RNA leaving one 5’ ribonucleotide adjacent to the DNA
What is telomeric DNA synthesised and maintained by?
Telomerase
What is mismatch repair?
Mismatched bases or loops are replaced
Absence causes high cancer rates
What is base excision repair?
Replaces bases lost through depurination or deamination
What does DNA Glycosylase do?
Identifies and removes damaged base leaving apurinic or apyrimidinic site
What does AP endonuclease do?
Cuts the backbone
What does Exonuclease do?
Removes sugar and several adjacent bases
What repairs the strands after mismatch repair and base excision repair?
DNA Polymerase
Ligase
What is nucleotide excision repair?
Responds to helix distortion: pyrimidines sticking together
Replaces regions of damaged DNA
Name 2 diseases caused by mutations in the nucleotide excision repair pathway
Cockayne syndrome
Xeroderma Pigmentosum
What is Xeroderma Pigmentosum caused by?
Lack of enzymes necessary for repair of DNA damage induced by UV radiation
Symptoms of XP?
Extreme sensitivity to sunlight
Skin cancer
Secondary tumours and cancer-related death
What is nonhomologous end joining?
Ku protein recognises double stranded breaks
Holds both strands of broken DNA leaving ends accessible to nucleases, polymerases and ligases
Ends of broken DNA aligned, filled and strands ligated
What happens in recombination repair?
Enzymes and proteins are used that perform genetic recombination between homologous chromosomes during meiosis
Uses DNA sequence information in the homologous chromosome to correct the break
