Lecture 2 - Exam 2 Flashcards
Discuss the vital prokaryotic DNA replication enzymes.
DNA pol III: Main enzyme in DNA replication. Synthesizes new strands by adding nucleotides to the 3’ - OH group of the RNA primer. 3’ - 5’ exonuclease activity allows proofreading of DNA strand. 1000 nt/s.
DNA pol I: Removes the RNA primer from Okazaki fragments by 5’-3’ exonuclease activity and fills the gap by adding nucleotides to the lagging strand fragments… It has both 5’-3’ and 3’-5’ exonuclease activity.
DNA pol II: Its main role is in repair and also a backup of DNA polymerase III. It has 3’-5’ exonuclease activity.
As replication in bacteria is bidirectional, the replication forks will….?
As replication in bacteria is bidirectional, at some point the two replication forks will meet (about 180 degrees from oriC).
As replication in bacteria is bidirectional, at some point the two replication forks will meet (about 180 degrees from oriC). This phase of replication is unique to?
This phase of replication is unique to termination and is defined as replication fork “convergence.”
Termination occurs in the…?
What does this place contain?
“Termination zone,” which contains multiple Ter sites and bind to Tus proteins on the replisome.
Replication forks can meet…?
Anywhere within the termination zone, either at a Ter site or between Ter sites.
Eventually, the two replicating forks will meet somewhere 180 degrees from the oriC, and will ____________ replication.
Terminate
There are multiple _________ sites (______ sites) on the bacterial chromosome. E. coli has how many?
Termination ; Ter
E. coli has 6.
What are Ter sites?
22-bp sequences that binds to specific proteins called TUS proteins (Termination Utilization Substance). Ter sites serve as binding sites for Tus protein. Ter sites only allow passing of the replication fork in one direction.
Ter sites are divided into?
Two groups of three (or more) termination sequences:
terA, terD, and terE prevent counterclockwise replication.
terC, terB, and terF prevent clockwise rotation.
These termination recognizing sequences (Ter sites) create a “_____” that the replication fork can enter, but can’t leave.
Trap
The one way travel is imposed at Ter sites by?
The Tus protein which bind to Ter sites and inhibit the helicase (DnaB) activity.
When Tus binds to Ter site, a complex is formed that locks the DNA strands together and prevents DnaB helicase from further separating the DNA.
What happens now after replication has been terminated? How is the replication of chromosomes completes? How are the two pieces of replicated DNA joined?
Completion of replication involves:
1) A short-term over-replication of the region where the replication forks converge.
2) The excess regions are then cut by RecBCD proteins, and the strands are joined together with DNA ligase.
Completion of replication therefore requires _____________ needed to repair _____________, part of the SOS response that is also used to repair DNA damage.
Several proteins (RecBCD) ;
double-strand DNA breaks
What are SbcC, SbcD, and Exol?
Highly conserved nucleases required for genomic stability-enzyme process DNA intermediates at sites where replication forks converge.
What does the process of overreplication of replication forks look like?
- Replication forks continue past their meeting point, creating a palindromic substrate.
- SbcCD-Exol cleave and process the overreplicated intermediate.
- RecBCD-mediated resection and joining of the DNA ends completes replication.
- In vitro and proposed in vivo substrates for SbcCD, respectively.
What are the models of DNA replication?
Factory model: DNA polymerase is stationary in the cell and the DNA is pulled through it (pulled through the polymerase).
Train on track model: DNA polymerase moves along the DNA strands like a train moving on the track.
More recent data tends to support which model of DNA replication?
Train on the track.
Under optimal growth conditions for E. coli, cell division occurs more rapidly than…?
What does this require?
Genome replication.
This requires a new round of genome replication to begin in the newly replicated chromosome before the previous round has been completed.
Under optimal growth conditions for E. coli, cell division occurs more rapidly than genome replication.
This requires a new round of genome replication to begin in the newly replicated chromosome before the previous round has been completed.
However, there must be only one…?
There must be only one replication per cell cycle.
To ensure that initiation at an oriC occurs only once per cell cycle, what happens?
Specific mechanisms exist to control chromosomal replication. This control is primarily mediated by DNA methylation.
DNA methylation is a _____________ that acts by the ______________ from an S-adenosyl-methionine molecule to an ______ or a ______ in the DNA.
Base modification system ;
Addition of a methyl group ;
Adenine ;
Cytosine
DNA methylation plays vital roles in…?
DNA replication and repair.
Enzymes responsible for DNA methylation are called?
DNA-methyltransferases (MTases)
“Mature” DNA is methylated (primarily) at the?
N6 positions in adenines found in the sequence 5’ GATC 3’.
Is there a time where DNA is not methylated?
Yes, there is a short amount of time when the newly synthesized DNA is NOT methylated. During this period, the DNA is referred to as hemimethylated, because the parent strand is methylated and the daughter strand is not.
There are ______ GATC sequences in the oriC locus, so this area initially has?
Multiple ;
This area initially has a lot of unmethylated DNA following replication.
What is the protein called that binds to the hemimethylated oriC DNA?
A protein called SeqA binds to this hemimethylated oriC DNA and “sequesters” it, delaying DNA methylation and recognition and binding of DnaA.
DNA damage is a common occurrence in all cells. A bacterial cell growing in an aerobic environment will suffer 3000-5000 DNA lesions per cell per generation. Most of this damage is repaired by…?
DNA repair mechanisms and do no impact replication.
This is the reason why the replication forks are not meeting perfectly at 180 degrees from oriC and meeting at the same time, because they will break down during replication, as DNA damage is common in cells.
When a DNA polymerase encounters an unrepaired DNA lesion, it is unable to do what?
It is unable to bypass the DNA damage and the DNA Pol breaks down and the replication fork stalls. Under normal cellular growth conditions, nearly every bacterial replication fork will suffer this fate.
What does a stalled replication fork trigger?
What is this process called?
An elaborate enzymatic response, ultimately restoring an active replication fork without introducing a genetic mutation. This process is called homologous recombination.
Homologous recombination repair mechanisms involve _____ enzymes and is _____.
Multiple ;
Complex
In a nutshell, what is homologous recombination?
Homologous recombination (HR) repair uses a complex “cross-over” mechanism to faithfully repair the damaged DNA. It uses the methylated strand to know where to go in and repair the DNA without a mutation.
Typically, homologous recombination yields…?
Two complete chromosome monomers when replication is terminated.
Sometimes there are unequal numbers of crossover events. What happens?
Sometimes, when there are unequal numbers of crossover events, homologous recombination causes the two replicated chromosomes to be connected, resulting in a dimeric genome (covalent chromosome dimers)…
At the end of replication, you won’t have two separate chromosomes, will have a big ass chromosome dimers because of unequal crossovers.
What is chromosome separation?
Detachment of the newly replicated chromosomes from one another.
