bacterial cell cycle and DNA replication Flashcards
The cell cycle of many bacterial species involves binary fission. Which steps are involved?
cell elongation while the chromosome is replicated and segregated, formation of septal ring, invagination and finally cell separation
What are the three main growth phases of bacterial populations?
lag phase
exponential phase
stationary phase
How many origins does the chromosome of the bacterium Escherichia coli contain and by how many replication forks is it replicated?
A single origin at which two replication forks are assembled ?
What is generally meant by the process of DNA replication?
The duplication of the genome using the parental DNA as a template
Where does DNA replication take place in an Escherichia coli cell?
In the cytoplasm ?
How is DNA replicated at a replication fork in Escherichia coli?
Both leading and lagging strand synthesis are discontinuous
What is the approximate speed of DNA replication in Escherichia coli?
Between 600 and 1000 nt/s
Which are the main enzymes involved in Okazaki fragment maturation in Escherichia coli?
DNA ligase and DNA polymerase I
How is binding of the main initiator protein DnaA at the origin able to initiate DNA synthesis?
binding of DnaA at so-called DnaA-boxes bends the DNA, which puts pressure on the AT-rich region and causes it to melt, allowing the recruitment of the replicative helicase
How can it be avoided that the origin of replication is initiated immediately after a first round of replication has started?
GATC sequences in the E. coli chromosome are normally fully methylated by Dam, but hemi-methylated after replication starts. Hemi-methylated DNA is bound by SeqA, which prevents DnaA from binding (a process called sequestration)
14 Where is termination of replication taking place in the Escherichia coli chromosome?
In a specialised termination zone opposite the origin
What are the three stages of chromosome replication in Escherichia coli?
Initiation, elongation and termination of DNA replication
Under optimal growth condition Escherichia coli can double every 20 min. How long does one full round of DNA replication take in E. coli?
40 min, as E. coli can have overlapping rounds of DNA synthesis
In the active centre of the replicative polymerase the next free base on the template strand is a G. Which nucleotide needs to be incorporated into the nascent strand?
dCTP
In the active centre of the replicative polymerase the next free base on the template strand is a T. Which nucleotide will diffuse into the active centre of the polymerase?
dTTP, dATP, dCTP, dGTP ?
In the active centre of the replicative polymerase the next free base on the template strand is a T. How is the correct nucleotide incorporated with high accuracy?
Only dATP is orientated in a way that the alpha-phosphate of the triphosphate can undergo a nucleophilic attack of the 3’-OH of the primer end
Which ion is an important co-factor in the active centre of polymerases?
Mg2+, it makes the alpha-phosphate more positive by drawing electrons away
How much does the addition of the replicative polymerase in Escherichia coli increase accuracy of DNA synthesis?
1000 fold
How much does the proof-reading activity of the replicative polymerase in Escherichia coli increase accuracy of DNA synthesis?
100 - 1000 fold ?
How much does methyl-directed mismatch repair (MMR) in Escherichia coli increase accuracy of DNA synthesis?
100 fold
What does the methyl-directed mismatch repair system (MMR) in Escherichia coli do?
It recognises mispaired bases in the double-strand, such as a G-T mispair
DNA Replication has left a G-T mispair behind, which is recognised by methyl-directed mismatch repair. How can MMR identify which is the wrongly incorporated base?
it uses hemi-methylated GATC sequences as a marker. MutH generates a nick in the un-methylated strand, as this is the newly synthesised strand
Why do replicative polymerases always need a primer, whereas other polymerases such as RNA polymerases do not?
The primer-template complex is held under tension in the active site of the polymerase via multiple interactions between amino acid residues and the DNA. The template strand alone would not be held in place but would flip out of the active site
How can a wrongly incorporated nucleotide be recognised and then excised by the proof reading activity of the replicative DNA polymerase in Escherichia coli?
The primer-template complex is held under tension in the active site of the polymerase via multiple interactions between amino acid residues and the DNA. When a wrong base is inserted the primer end is not held in place and flips into the exonuclease site
