bacterial cell cycle and DNA replication

Question 1

The cell cycle of many bacterial species involves binary fission. Which steps are involved?

Answer 1

cell elongation while the chromosome is replicated and segregated, formation of septal ring, invagination and finally cell separation

Question 1

What are the three main growth phases of bacterial populations?

Answer 1

lag phase
exponential phase
stationary phase

Question 2

How many origins does the chromosome of the bacterium Escherichia coli contain and by how many replication forks is it replicated?

Answer 2

A single origin at which two replication forks are assembled ?

Question 3

What is generally meant by the process of DNA replication?

Answer 3

The duplication of the genome using the parental DNA as a template

Question 4

Where does DNA replication take place in an Escherichia coli cell?

Answer 4

In the cytoplasm ?

Question 5

How is DNA replicated at a replication fork in Escherichia coli?

Answer 5

Both leading and lagging strand synthesis are discontinuous

Question 6

What is the approximate speed of DNA replication in Escherichia coli?

Answer 6

Between 600 and 1000 nt/s

Question 7

Which are the main enzymes involved in Okazaki fragment maturation in Escherichia coli?

Answer 7

DNA ligase and DNA polymerase I

Question 8

How is binding of the main initiator protein DnaA at the origin able to initiate DNA synthesis?

Answer 8

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

Question 9

How can it be avoided that the origin of replication is initiated immediately after a first round of replication has started?

Answer 9

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)

Question 10

14 Where is termination of replication taking place in the Escherichia coli chromosome?

Answer 10

In a specialised termination zone opposite the origin

Question 11

What are the three stages of chromosome replication in Escherichia coli?

Answer 11

Initiation, elongation and termination of DNA replication

Question 12

Under optimal growth condition Escherichia coli can double every 20 min. How long does one full round of DNA replication take in E. coli?

Answer 12

40 min, as E. coli can have overlapping rounds of DNA synthesis

Question 13

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?

Answer 13

dCTP

Question 14

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?

Answer 14

dTTP, dATP, dCTP, dGTP ?

Question 15

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?

Answer 15

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

Question 16

Which ion is an important co-factor in the active centre of polymerases?

Answer 16

Mg2+, it makes the alpha-phosphate more positive by drawing electrons away

Question 17

How much does the addition of the replicative polymerase in Escherichia coli increase accuracy of DNA synthesis?

Answer 17

1000 fold

Question 18

How much does the proof-reading activity of the replicative polymerase in Escherichia coli increase accuracy of DNA synthesis?

Answer 18

100 - 1000 fold ?

Question 19

How much does methyl-directed mismatch repair (MMR) in Escherichia coli increase accuracy of DNA synthesis?

Answer 19

100 fold

Question 20

What does the methyl-directed mismatch repair system (MMR) in Escherichia coli do?

Answer 20

It recognises mispaired bases in the double-strand, such as a G-T mispair

Question 21

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?

Answer 21

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

Question 22

Why do replicative polymerases always need a primer, whereas other polymerases such as RNA polymerases do not?

Answer 22

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

Question 23

How can a wrongly incorporated nucleotide be recognised and then excised by the proof reading activity of the replicative DNA polymerase in Escherichia coli?

Answer 23

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