Unit 3: DNA REPLICATION AND EXTRACHROMOSOMAL REPLICATION Flashcards
Topoisomerase
An enzyme that changes the number of times the two strands in a closed DNA molecule cross each other.
It does this by cutting the DNA, passing DNA through the break, and resealing the DNA
-it also Allows chromosome to be relaxed
Replisome
A multi-protein structure that assembles at the bacterial replication fork to undertake synthesis of DNA Replisomes do not exist as independent units and contain DNA polymerase and other enzymes. As the replisome moves along DNA, the parental strands unwind and daughter strands synthesize. Replisome synthesizes the daughter strand
Proteins bind to origin –> separate DNA strand –> DNAPoly and other proteins go into replisome –>synthesize daughter DNA
Conditional lethal
a mutation that is lethal under one set of conditions but is not lethal under a second set of conditions such as temperature. Conditional lethals are able to accomplish replication under permissive conditions typically provided by the normal temperature of incubation) but they are defective under non-permissive or restrictive conditions (provided by the higher temperature of 42C)
What is in vitro complementation used for?
A functional assay used to identify components of a process (in this case replication apparatus)
The reaction is reconstructed using extracts from a mutant cell aka an in vitro system for replication is prepared from a dna mutant and is operated under conditions in which the mutant gene is inactive
Fractions from wild-type cells are then tested for restoration of activity
Dna Mutants
Temperature-sensitive replication mutants that are defective in replication elongation during synthesis of DNA
Distinguish two stages of replication by their behavior when the temperature is raised
1) Quick-stop mutants
2) Slow-stop mutants
Quick-stop mutants
cease replication immediately upon a temperature increase. Defective in the components of the replication apparatus, typically in the enzyme needed for ELONGATION
Slow-stop mutants
temperature-sensitive replication mutants that are defective in INITIATION of replication. Slow-stop mutants complete the current round of replication but cannot INITIATE another round. Hence defective in initiation of new cycle of replication at the origin
DnaA-ATP and the prepriming complex bind to the origin of replication and do what?
start separating DNA (initiation of replication)
Synthesis of DNA occurs both during what two events?
-replication and DNA repair
What occurs in repair reactions?
the damage nucleotide and other bases are removed from the damaged strand and DNA polymerase comes in and synthesizes complementary strand accurately getting rid of damaged base
An enzyme that can synthesize a new DNA strand on a template strand is called what?
DNA polymerase
an enzyme that undertakes semiconservative replication (not involved in repair) is called a?
DNA replicase
Fill in the blank: DNA is synthesized by adding new nucleotides to the ____ end of the growing chain/strand
3’-OH
The incoming nucleotide that is added to the primer 3’-OH is a ? and what is released when the nucleotide is added to the growing chain?
5’ triphosphate
-diphosphate is released when nucleotide is added
DNA grows in what direction?
5’ to 3’ direction
In bacteria, which polymerase is involved in replication?
DNA polymerase III; responsible for synthesis of new strands of DNA
What is holoenzyme?
a large protein structure of replicases
What is the role of DNA poly II?
required to restart replication fork when its progressed is blocked by damage in DNA
DNA polymerase IV and DNA polymerase IV are both involved in what?
translesion replication. Involved in allowing replication to bypass certain types of damage and are called error-prone polymerases
What is meant by semiconservative replication?
The two strand of parental duplex are separated and each serve as a template for synthesis of daughter strand. Thus, there will be two daughter duplexes, each of which has one parental strand and one newly synthesized strand
What is a DNA repair reaction?
Repair synthesis replaces a short stretch of one strand of DNA containing a damaged base.
One strand of DNA is damaged. It is excised and new material is synthesized to replace it.
All prokaryotic and eukaryotic DNA polymerases share the same fundamental type of synthesis activity. What is it?
- antiparallel synthesis from 5’ to 3’ from a template that is 3’ to 5’
- this means adding nucleotides one at a time to a 3’-hydroxyl (OH) growing end meaning chain grows in the 5’-3’ direction
- the choice of the nucleotide to add to the chain is dictated by base pairing with the complementary template strand
What is the unique role of DNA polymerase I?
DNA poly I has a unique 5’-3’ exonuclease activity. The exonuclease activity is also used to excise bases that have been added to DNA incorrectly. This provides a proofreading error control system. This exonuclease activity can be combined with DNA synthesis to perform nick translation.
What happens to DNA pol I when treated with protease? What are the subunits of Pol I?
DNA pol I cleaves into two subunits when treated w/a protease. The large subunit is called the cleanout fragment and the smallest subunit has the 5’-3’ exonuclease activity which removes about 10 bases at a time.