Lecture 5 - chromosomes and DNA replication Flashcards
Summary of DNA structure
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
Significance of DNA grooves and fluorescence microscopy
- Lots of hydrophobic interaction between stacked layers; allows DAPI and acridine orange enter grooves in DNA to fluoresce.
Major groove and DNA binding proteins
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.
How do DNA proteins have sequence specificity?
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
Structure of the bacterial nucleoid
Contains topologically separated domains anchored by histone-like proteins
Purpose of histone-like proteins in the nucleoid
Anchoring proteins prevent relaxation of one region of DNA from spreading to the rest of the chromosome (maintains compaction of DNA)
What does supercoiling do for the overall energy state of DNA?
Both positive and negative supercoiling have a higher energy state than relaxed DNA.
Positive vs negative supercoiling
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
What organisms possess positively supercoiled DNA?
Archaea living in acid at high temperatures; makes them more resistant to DNA degradation
What organisms possess negatively sueprcoiled DNA?
Eukaryotes, bacteria, and most archaea; makes it easier to open DNA up for reading
Function of topoisomerases
Modulate level of genomic supercoiling
Type I Topoisomerases
- usually single subunit enzymes
- cleave and reseal only one strand of DNA, therefore can only relieve supercoils (can’t increase supercoiling)
Type II Topoisomerases
- multi subunit enzymes
- cleaves both strands of DNA and uses ATP hydrolysis to change supercoiling
DNA Gyrase
Type II topoisomerase that is targeted by aminocoumarin and quinolone antibiotics
How does topoisomerase I work?
Cleaves one strand of the double helix and passes the other strand through to remove one supercoil
How does DNA gyrase work?
Cleaves both strands of the double helix and passes the remaining intact double stranded section through to add one supercoil.
How does novobiocin inhibit DNA gyrase activty?
Competitively binds GyrB (subunit of DNA gyrase) and prevents ATP from binding
How do cipro and nalidixic acid inhibit DNA gyrase activity?
Prevent re-ligation of double stranded breaks
Why did we move from nalidixic acid to cipro?
It was too easy for bacteria to become resistant to nalidixic acid
Purpose of DnaA
Controls initiation of DNA replication. Accumulates during cell growth and triggers replication at specific sites near the origin of replication.
How does DnaA work?
DnaA-ATP complexes bind to 9-bp repeats upstream of the origin –> looping in DNA to prepare for strand separation by DNA helicase (DnaB)
What is the purpose of the clamping protein in DNA polymerase III?
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
Purpose of RNA polymerase
Places RNA primer for initiation of DNA replication
How are RNA primers removed during replication?
- RNase H recognizes the RNA-DNA mismatch and cleaves the RNA.
- DNA Pol I fills the gap with DNA
- DNA ligase repairs the nick
How does Tus work in termination of replication
Binds terminator (ter) sequences and acts as a counter-helicase to stop the progress of DNA helicase
How are linked (catenated) chromosomes separated at the end of replication?
Topoisomerase IV or XerC and XerD resolvases
How are ter sequences distributed on the chromosome?
Along the ter macrodomain, some stop clockwise replication and some stop counterclockwise rotation. Multiple ter sequences for both directions to ensure that replication stops
Mechanism of daughter chromosome deacatenation
- dif (deletion induced filamentation) sequence at the Ter macrodomain is recognized by XerC
- If two dif sequences are nearby (as in the case of linked chromosomes), XerC will bind both and form a Holliday junction
- XerD resolves the Holliday junction and separates the chromosomes
Plasmids
Extragenomic, autonomously replicating DNA molecules.
- usually circular and sometimes much smaller than chromosomes
- require host proteins to replicate
Copy number of plasmids in bacterial cells
Can range from 1 to 1000.
Copy number vs plasmid size
Plasmid production exerts metabolic drag on the host cell. The bigger the plasmid, the lower the copy number
Problems with low copy number plasmids
- Need machinery to ensure that plasmids are properly partitioned into daughter cells
- can more easily be lost by the host cell
What advantages can plasmids give a bacteria?
Occupation of different niches
- Resistance to antibiotics/toxic metals
- pathogenesis
- symbiosis
Methods of plasmid replication
- Bidirectional (“theta”) replication
- Rolling circle replication
How does bidirectional (“theta”) replication work?
Starts at a single origin and occurs in two directions simultaneously; same as chromosomal replication
How does rolling circle replication work?
Starts at a single origin and moves in one direction.
Steps of rolling circle replication
- Rep dimer makes a nick on one strand and binds to the 5’ end
- 3’ end of the nick is extended by DNA pol III
- single strand DNA on outer strand is displaced as new DNA is made
- Rep releases circularized old outer strand after full replication of outer strand
- Old outer strand receives a complementary inner strand
Mechanism of plasmid partitioning
- ParR binds plasmid and ParM
- ParM-ATP monomers bind ParR and form filaments that push one another away
- Plasmids are pushed apart
What is ParM
Structural homolog of actin that forms filament for plasmid partitioning
Purpose of toxin-antitoxin modules
Used in post-segregational killing to kill daughter cells that don’t receive the plasmid after segregation
How does toxin-antitoxin work in post-segregational killing
- Toxin and antitoxin production are driven by the same promoter –> produce a dimer of toxin and antitoxin
- Toxin is held inactive when bound to antitoxin
- Antitoxin is degraded overtime by proteases
- If there is no plasmid to continue production of antitoxin molecules, the cell will die
How does a cell die in post-segregational killing?
Toxin poisons DNA gyrase and prevents re-ligation of double stranded breaks
