Lecture 5 - chromosomes and DNA replication

Question 1

Summary of DNA structure

Answer 1

Polymer of nucleotides connected by phosphodiester bonds. Two strands of DNA are oriented in an antiparallel manner with purines of one strand bonding to pyrimidines of the other

Question 2

Significance of DNA grooves and fluorescence microscopy

Answer 2

• Lots of hydrophobic interaction between stacked layers; allows DAPI and acridine orange enter grooves in DNA to fluoresce.

Question 3

Major groove and DNA binding proteins

Answer 3

Provides sequence info for binding proteins. Binding proteins are able to read out the sequence of nucleotides based on the pattern of H donors/acceptors.

Question 4

How do DNA proteins have sequence specificity?

Answer 4

Insertion into the major groove with the correct sequence. If there are multiple binding sites, these are usually about 10 base pairs apart to account for the natural spiral of DNA

Question 5

Structure of the bacterial nucleoid

Answer 5

Contains topologically separated domains anchored by histone-like proteins

Question 6

Purpose of histone-like proteins in the nucleoid

Answer 6

Anchoring proteins prevent relaxation of one region of DNA from spreading to the rest of the chromosome (maintains compaction of DNA)

Question 7

What does supercoiling do for the overall energy state of DNA?

Answer 7

Both positive and negative supercoiling have a higher energy state than relaxed DNA.

Question 8

Positive vs negative supercoiling

Answer 8

Positive: result from over-winding of DNA (adding an extra twist of the helix) and makes the strands harder to separate
Negative: results from under-winding of DNA (taking out one twist of the helix) and makes the strands easier to separate

Question 9

What organisms possess positively supercoiled DNA?

Answer 9

Archaea living in acid at high temperatures; makes them more resistant to DNA degradation

Question 10

What organisms possess negatively sueprcoiled DNA?

Answer 10

Eukaryotes, bacteria, and most archaea; makes it easier to open DNA up for reading

Question 11

Function of topoisomerases

Answer 11

Modulate level of genomic supercoiling

Question 12

Type I Topoisomerases

Answer 12

• usually single subunit enzymes
• cleave and reseal only one strand of DNA, therefore can only relieve supercoils (can’t increase supercoiling)

Question 13

Type II Topoisomerases

Answer 13

• multi subunit enzymes
• cleaves both strands of DNA and uses ATP hydrolysis to change supercoiling

Question 14

DNA Gyrase

Answer 14

Type II topoisomerase that is targeted by aminocoumarin and quinolone antibiotics

Question 15

How does topoisomerase I work?

Answer 15

Cleaves one strand of the double helix and passes the other strand through to remove one supercoil

Question 16

How does DNA gyrase work?

Answer 16

Cleaves both strands of the double helix and passes the remaining intact double stranded section through to add one supercoil.

Question 17

How does novobiocin inhibit DNA gyrase activty?

Answer 17

Competitively binds GyrB (subunit of DNA gyrase) and prevents ATP from binding

Question 18

How do cipro and nalidixic acid inhibit DNA gyrase activity?

Answer 18

Prevent re-ligation of double stranded breaks

Question 19

Why did we move from nalidixic acid to cipro?

Answer 19

It was too easy for bacteria to become resistant to nalidixic acid

Question 20

Purpose of DnaA

Answer 20

Controls initiation of DNA replication. Accumulates during cell growth and triggers replication at specific sites near the origin of replication.

Question 21

How does DnaA work?

Answer 21

DnaA-ATP complexes bind to 9-bp repeats upstream of the origin –> looping in DNA to prepare for strand separation by DNA helicase (DnaB)

Question 22

What is the purpose of the clamping protein in DNA polymerase III?

Answer 22

Ensures that DNA Pol III can stay on the DNA for the entirety of replication. Otherwise, DNA Pol would fall off and have to be re-loaded onto the strand

Question 23

Purpose of RNA polymerase

Answer 23

Places RNA primer for initiation of DNA replication

Question 24

How are RNA primers removed during replication?

Answer 24

1. RNase H recognizes the RNA-DNA mismatch and cleaves the RNA.
2. DNA Pol I fills the gap with DNA
3. DNA ligase repairs the nick

Question 25

How does Tus work in termination of replication

Answer 25

Binds terminator (ter) sequences and acts as a counter-helicase to stop the progress of DNA helicase

Question 26

How are linked (catenated) chromosomes separated at the end of replication?

Answer 26

Topoisomerase IV or XerC and XerD resolvases

Question 27

How are ter sequences distributed on the chromosome?

Answer 27

Along the ter macrodomain, some stop clockwise replication and some stop counterclockwise rotation. Multiple ter sequences for both directions to ensure that replication stops

Question 28

Mechanism of daughter chromosome deacatenation

Answer 28

1. dif (deletion induced filamentation) sequence at the Ter macrodomain is recognized by XerC 2. If two dif sequences are nearby (as in the case of linked chromosomes), XerC will bind both and form a Holliday junction 3. XerD resolves the Holliday junction and separates the chromosomes

Question 29

Plasmids

Answer 29

Extragenomic, autonomously replicating DNA molecules. - usually circular and sometimes much smaller than chromosomes - require host proteins to replicate

Question 30

Copy number of plasmids in bacterial cells

Answer 30

Can range from 1 to 1000.

Question 31

Copy number vs plasmid size

Answer 31

Plasmid production exerts metabolic drag on the host cell. The bigger the plasmid, the lower the copy number

Question 32

Problems with low copy number plasmids

Answer 32

- Need machinery to ensure that plasmids are properly partitioned into daughter cells - can more easily be lost by the host cell

Question 33

What advantages can plasmids give a bacteria?

Answer 33

Occupation of different niches - Resistance to antibiotics/toxic metals - pathogenesis - symbiosis

Question 34

Methods of plasmid replication

Answer 34

1. Bidirectional ("theta") replication 2. Rolling circle replication

Question 35

How does bidirectional ("theta") replication work?

Answer 35

Starts at a single origin and occurs in two directions simultaneously; same as chromosomal replication

Question 36

How does rolling circle replication work?

Answer 36

Starts at a single origin and moves in one direction.

Question 37

Steps of rolling circle replication

Answer 37

1. Rep dimer makes a nick on one strand and binds to the 5' end 2. 3' end of the nick is extended by DNA pol III 3. single strand DNA on outer strand is displaced as new DNA is made 4. Rep releases circularized old outer strand after full replication of outer strand 5. Old outer strand receives a complementary inner strand

Question 38

Mechanism of plasmid partitioning

Answer 38

1. ParR binds plasmid and ParM 2. ParM-ATP monomers bind ParR and form filaments that push one another away 3. Plasmids are pushed apart

Question 39

What is ParM

Answer 39

Structural homolog of actin that forms filament for plasmid partitioning

Question 40

Purpose of toxin-antitoxin modules

Answer 40

Used in post-segregational killing to kill daughter cells that don't receive the plasmid after segregation

Question 41

How does toxin-antitoxin work in post-segregational killing

Answer 41

1. Toxin and antitoxin production are driven by the same promoter --> produce a dimer of toxin and antitoxin 2. Toxin is held inactive when bound to antitoxin 3. Antitoxin is degraded overtime by proteases 4. If there is no plasmid to continue production of antitoxin molecules, the cell will die

Question 42

How does a cell die in post-segregational killing?

Answer 42

Toxin poisons DNA gyrase and prevents re-ligation of double stranded breaks