Replication Process (Prokaryote)_Lec4 Flashcards
Replication - Definition and phases
short
An enzymatic process performed by the DNA Pol protein complex
5 ->3
- Initiation
- recognition of oriC
- separation of the two DNA strands and their stabilisation in the single-stranded state
- PRIMOSOME (Protein complex) - Elongation
- involves physical bi-directional duplication of DNA (Replication) from the oriC to the terC
- REPLISOME (Protein complex): DNA Pol (1, 2 and 3)
DNA Polymerase I, II and III (short)
DNA Pol I
- helps with the repair of existing DNA
- show the highest polymerase activity , although DNA Pol III is responsible for replication
DNA Pol II
- DNA repair
DNA Pol III
- responsible for the main polymerase rxn during replication
DNA Pol I - Structure : composition - Enzymatic activity : 5' - 3' extension 3'- 5' exonuclease 5' - 3' exonuclease - Mutants Mutant phenotype Lethality
Structure : composition - Monomeric
Enzymatic activity :
5’ - 3’ extension - Yes
3’- 5’ exonuclease - Yes
5’ - 3’ exonuclease - Yes
Mutants
Mutant phenotype - Defective for repair
Lethality- Vitality declines when 5’ - 3’ exonuclease activity is affected
DNA Polymerase II - Structure : composition - Enzymatic activity : 5' - 3' extension 3'- 5' exonuclease 5' - 3' exonuclease - Mutants Mutant phenotype Lethality
Structure : composition - Not known
Enzymatic activity :
5’ - 3’ extension - Yes
3’- 5’ exonuclease - Yes
5’ - 3’ exonuclease - No
Mutants:
Mutant phenotype - No effect
Lethality - No effect
DNA Polymerase III
- Structure : composition
- Enzymatic activity :
5’ - 3’ extension
3’- 5’ exonuclease
5’ - 3’ exonuclease
Structure : composition - Hetero-multimeric
Enzymatic activity :
5’ - 3’ extension - Yes
3’- 5’ exonuclease - Yes
5’ - 3’ exonuclease - No
DNA Polymerase III
- Structure : composition
- Enzymatic activity :
5’ - 3’ extension
3’- 5’ exonuclease
5’ - 3’ exonuclease - Mutants
Mutant phenotype
Lethality
Structure : composition - Hetero-multimeric
Enzymatic activity :
5’ - 3’ extension - Yes
3’- 5’ exonuclease - Yes
5’ - 3’ exonuclease - No
Mutants
Mutant phenotype - prevents replication
Lethality - Conditional lethal ( essential for growth)
DNA Polymerase unique properties not found in RNA polymerase
5’ - 3’ polymerase activity
NB: 3’ - 5’ and 5’ - 3’ exonuclease activities
* allows replisome to proofread
Proofread function of the replisome is controlled on two levels
- Error control before synthesis
- bases are carefully examined before DNA synthesis - Error control after synthesis
- bases in dsDNA are carefully examined after DNA synthesis in order to detect mistakes
Subunits of DNA Pol III and their function`
- alpha - DNA SYNTHESIS
- Beta - none
- 0 with a line through the middle - none
- e - 3’ - 5’ exonulcease
- gamma ( i think) - processivity of DNA
- Y - Processivity of DNA
- looks like a t - Processivity of DNA
DNA Synthesis : Replication process - Two activities
- Continuous DNA synthesis
- 5’ -> 3’ direction
- leading strand = templaye - Discontinuous DNA synthesis
- 3’ -> 5’ on lagging strand
- Okazaki fragments
short fragments synthesized in the 5’-> 3’ direction
Proteins (12) required during DNA replication + function- Prokaryotes
DnaA: Activates replication initiation, helps unwind
DnaB - Helicae : Separate DNA strands
DnaC: Helps with binding of the helicase
HU: Helps DnaA during initiation of replication, regulates replication process
SSB: Stabilise single stranded DNA
Gyrase: Topoisomerase, maintains negative supercoiling, relieves tension due to unwinding
subunits: A and B
- GyrA: DNA breakage and repair
- GyrB: ATP hydrolysis for energy transduction
DnaG - primase: synthesis of RNA primers
DNA Pol III Holoenzyme: DNA synthesis
DNA Pol I: proofreading
RNase H: Removes RNA primers
DNA ligase: ligates okazaki fragments together
Steps in DNA synthesis (10)
look at notes
Primosome - proteins
Helicase
Gyrase
SSB
Primase
