Lecture 8 Genomics and Replication

Question 1

How do microbes supercoil?

Answer 1

They introduce double strand breaks, then reseal. It helps pack the DNA, using many proteins.

Question 2

Positive supercoiling

Answer 2

DNA is overwound.

Question 3

Negative supercoiling

Answer 3

DNA is underwound. Everything usually uses this type of super coiling, except Archea that live in acid and high temperatures.

Question 4

What are Topoisomerases?

Answer 4

Enzymes that change DNA supercoiling. They don’t change the substrate, they just change the topo.
There are two Types of topoisomerases.

Question 5

Type I Topoisomerases

Answer 5

Usually single proteins
Cleaves one strand of DNA
Start out with 5 negative super coils. 3 helical turns to 4 helical turns. The Helix then winds in this region cleaved resulting one less negative supercoil, 4.

Question 6

Type II Topoisomerases

Answer 6

Multiple subunits
Cleaves both strands of DNA. Breaking the strand then gluing them back together.
E.g. DNA gyrase, targeted by quinolone antibiotics
inducing supercoiling in bacteria and also undoes supercoiling in bacteria.

Question 7

Describe the mechanism of DNA replication.

Answer 7

Replication starts at a single origin (oriC).
Replication bubble forms after initiation.
Two replication forks that move in opposite directions around the chromosome.
Ends at termination sites located on opposite to the origin.

Question 8

What are the major proteins involved in DNA Replication? (There are 6)

Answer 8

DnaA: Initiator protein
DnaB: Helicase
DNA primase: synthesis of the RNA primer
DNA pol III: major replication enzyme
DNA pol I: replaces the RNA primer with DNA
DNA gyrase: relieves DNA supercoiling

Question 9

What protein triggers the initiation of replication in E. coli?

Answer 9

DnaA; it accumulates during growth

Question 10

Initiation

Answer 10

The start of DNA replication is precisely timed and

linked to the ratio of DNA to cell mass.

Question 11

What is the sliding clamp protein used for?

Answer 11

The sliding clamp protein tethers the DNA pol to the DNA. With out it, the DNA pol would fall off the DNA molecule.

Question 12

Elongation of replicating DNA:

After initiation, each replication fork contains what two strands?

Answer 12

Leading and lagging strand.
Leading strand replicates in a 5’ to 3’ direction.
Lagging strand is replicated discontinuously, producing Okazaki Fragments.

Question 13

The cell coordinates the activity of two DNA Pol III
enzymes in one complex. These two enzymes, together with DNA primase and helicase, form the replisome. What is the replisomes job.

Answer 13

The replisome is to ensure the leading and lagging strands are synthesized in a 5’ to 3’ direction

Question 14

To remove RNA primers, use:

Answer 14

RNase H

Question 15

Termination Replication (ter)
Other words, what tells the polymerase to stop?
Use E. coli to explain.

Answer 15

The E. coli chromosome has 10 terminating sequences that polymers enter but rarely leave. One set of terminators deals with clockwise-replicating polymerase, wile the other set stops the DNA polymerase replicating counterclockwise relative to the origin.
Tus (terminus utilization substance) binds to these sequences and acts as a counter helicase.

Question 16

After DNA polymerase completes the duplication of the chromosome and has removed the ter sites, the cell is still face with a knotty problem. Because of the topology of the chromosome, the two daughter molecules will appear what way?

Answer 16

as catene, linked rings

Question 17

Describe mechanisms of genome evolution and

why plasmids are maintained in a population.

Answer 17

Plasmids have the ability to be transferred between cells. The have the advantage under certain conditions to be resistant to antibiotics and toxic metals. They can result pathogenesis, and symbiosis. They also have the ability to replicate two different ways: bidirectional replication and rolling-circle replication.
Evolution of a genome is a random process driven by
natural selection. Horizontal gene transfer and duplication followed by functional divergence through mutation contribute to genome reconstruction.

Question 18

bidirectional replication

Answer 18

This type of replication with a plasmid starts at a single origin and goes two directions simultaneously.

Question 19

rolling-circle replication

Answer 19

This type of replication with a plasmid starts at a single origin and moves in only one direction.

Question 20

Genomic islands

Answer 20

Provide evidence for horizontal gene transfer. Including pathogenicity islands, symbiosis islands, and fitness islands.

Question 21

Which of the following is NOT a reason plasmids have a high probability of being present in the next generation of cells?

a. Being present at a high number so some copies end up in each new cell after cell division
b. Encoding traits such as antibiotic resistance
c. Using actin-like ParM filament to bind to ParR-parC complexes on the plasmid
d. Carrying host survival genes
e. All of these ensure plasmids are passed on

Answer 21

e. all of these ensure plasmids are passed on