DNA Replication Flashcards
What two things are DNA replication considered?
Bidirectional
Semiconservative
Bidirectional DNA replication
Replication begins in the interior of a DNA molecule and proceeds in both directions
Semiconservative DNA replication
Each copy of the DNA molecule, after replication, contains one strand from the original template and one newly synthesized strand
Old strand plus a new strand
Origin of replication in prokaryotes
One origin
Circular DNA
Origin of replication in eukaryotes
- bidirectional
- multiple origins of replication, in order to replicate in a reasonable amount of time
Separation of the two complementary DNA strands in prokaryotes
- origin of replication needs to be melted (site where 2 strands separate
- origin of replication sequences are usually almost exclusively composed of A-T bases
Formation of the replication fork in prokaryotic DNA
- single stranded binding proteins (SSBs: bind to single strands to prevent reannealing and protect DNA from nuclease degradation
- DNA helicases then move toward the double stranded region and force the strands apart
- SSBs bind the newly separated strands
SSBs
Bind to single strands to prevent reannealing and protect DNA from nuclease degradation
DNA helicases
Forces apart the strands at the replication fork
-use ATP
Supercoiling
DNA is a helix, so when helicases separate the strands of DNA, supercoiling ahead of the replication fork will occur
Topoisomerases
Alleviate supercoiling ahead of the replication fork
Type I topoisomerase
Creates a nick in ONE strand which allows the DNA to swivel around the intact strand, then seals the nicked strand
Type II topoisomerases
Cut BOTH strands to relieve the supercoil, then re-legates the two strands
DNA gyrase
- special type II topoisomerase
- introduces negative supercoils
- also important for the separation of circular chromosomes after replication
- important for replication, packaging of the chromosome, and separation of replicated, circular chromosomes
What do quinolones inhibit??
DNA gyrase
-inhibits it so prokaryotes cannot replicate DNA
Too much quinolone
Can be toxic because it can inhibit mitochondrial DNA in eukaryotes
Direction of DNA replication
All plymerases that synthesize nuclei acids only catalyze synthesis in the 5’ to 3’ direction
What direction does the DNA template strand read?
3’ to 5’
LEading strand
At each replication fork, one strand of DAN fragment can be replicated continuously as the replication fork advances
Lagging strand
- synthesized discontinuously
- as the replication fork advances, small fragments of DNA are synthesized 5’ to 3’ away from the replication fork
- the fragments are called Okazaki fragments
Okazaki fragments
The discontinuously synthesized fragments.
Later joined to become a continuous segment of DNA
-combo of DNA and RNA
DNA polymerase
Require free 3’ OH group to being synthesis
-begin synthesis from the free 3’ OH group from the RNA primer after primase copies the first 10 nucleotide
Primase
an RNA polymerase that copies the first ~10 nucleotides to “prime” synthesis
-does not require free 3’ OH
RNA primer
Each new DNA fragment on the lagging strand begins with the action of primase laying down an RNA primer
DNA polymerase catalyze..
A reaction between the 3’ OH group of the strand being synthesized, and the 5’-triphsophate of an incoming nucleotide specified by the template being copied
Net reaction of DNA polymerase
Addition of a nucleotide to a growing DNA strand and the release of pyrophosphate
What makes the reaction of DNA polymerase irreversible?
Pyrophosphate
-it is further cleaved to inorganic phosphate to make the reaction irreversible and drive th reaction in the forward direction
What drivers the reaction that DNA catalyzes in the forward reaction?
Pyrophosphate
What does pyrophosphate do to the reaction catalyzes by DNA polymerase?
Coupled irreversible reaction
Coupled irreversible reaction
A common theme in many condensation reactions in biochemistry
-two high energy bonds are cleaved for each added nucleotide in a growing DNA chain
DNA polymerase III (pol III)
Enzyme in prokaryotes that elongates the leading and lagging strand
what elongates both the leading and lagging strand in prokaryotes?
DNA polymerase IIII
Proofreading
DNA replication needs to as accurate as possible, a single nucleotide mutation can have devastating consequences
What DNA polymerase has proofreading ability?
POL III (as well as many other DNA polymerases)
Pol III proofreading
Checks each added nucleotide to make sure it is correctly base-paired with the template strand
-exonuclease in reverse direction, 3’-5’ activity
What happens when Pol III detects a mistake?
Shifts backward one nucleotide and excises the misincorporated nucleotide
-exonuclease activity 3’-5’
3’-5’ exonuclease activity
Pol III shifts backward one nucleotide and excises the misincorporated nucleotide
What must happen to complete replication of circular DNA and the joining of Okazaki fragments?
The RNA primer must be removed and replaced with dNTPs (via Pol I)
DNA polymerase I (pol I)
- 5’-3’ polymerase activity
- 3’-5’ AND 5’-3’ exonuclease activity
What has BOTH 3’-5’ and 5’-3’ exonuclease activity?
DNA polymerase I
What all does Pol I do?
- removes RNA primer (5’ to 3’ exonuclease)
- replaces the rNTPs with the correct dNTP (5’-3’ polymerase)
- proof reads and corrects misincorporated nucleotides (3’-5’ exonuclease)
What can Pol I’s 5’-3’ exonuclease activity do?
Actually remove incorrectly base paired nucleotides (in this case the RNA primer)
-also important for another pol I function, DNA repair
What are the two important functions of Pol I 5’-3’ exonuclease activity?
- Replication of DNA
2. Repair of DNA
DNA ligase
Seals the nick that remains after the RNA primer is removed and replaced with dNTPs
DnaA protein
Initial strand separation
High A-T content
What prevents reannealing?
SSB proteins
G1 phase
Most variable in terms of time; growth and metabolism
G0 phase
Semipermanent G1 phase, appropriate signals can cause a re-entry into the cell cycle
S Phase
Replication of DNA (synthesis)
M phase
The stage of cell division (mitosis)
Pol alpha
Contains primase + DNA polymerase (begins strand synthesis)
Pol delta
DNA polymerase + proofreading (extends strands)
Pol beta and Pol epsilon
DNA repair enzymes
Pol gamma
Mitochondrial DNA polymerase
What happens to the lagging strand on the eukaryotic linear chromosome?
Will have a gap once the primer is removed
-overhang that is susceptible to degradation and has enzymes to properly manage them
Telomerase
Extends the ends of linear chromosomes (at the overhang)
Telomeres
The ends of eukaryotic chromosomes contain repeated sequences called this
-6-nucleotide repeats (>1000 6-nucleotide repeats)
What does telomerase contain?
- Segment of RNA that is complimentary to the telomere repeat and extends beyond the repeat, the extension acts a template
- also contains reverse transcriptase
Telomerase reverse transcriptase
Copies its own template (RNA) into DNA extending the 3’ overhang on the chromosome
- repeated many times
- keeps nucleases from chopping into important coding regions on end
After telomerase extends the repeat many times…
The overhang is filled in by the action of primase and DNA polymerase
-however, there will always be a section of DNA left that is single stranded
Section of DNA left that is single stranded
Assumes a special structure with the dsDNA and certain proteins to protect the end of the DNA
Is telomerase expressed in every cell?
No
Where is telomerase expressed?
In cells that continually divide and are not terminally differentiated
Cells that do not express telomerase
Have their chromosomes shortened at each cell division-have a finite number of cell divisions, cellular equivalent to aging
What causes cellular aging?
Cells that do not have telomerase have their chromosomes shorten every time there is replication, there is a finite number of cell divisions
Molecular reason behind cancer
Some cells can activate telomerase
What polymerases can copy RNA into DNA?
- Reverse transcriptase
- RNA dependent DNA polymerase
- telomerase
Common strategy in many viruses (HIV)
Lacks proofreading ability , high mutation rate
Strand-directed mismatch repair
Corrects errors made during replication
Damage repair
Similar process as strand-directed mismatch
DNA repair proteins
- With endonuclease activity recognize misincorporated bases or damaged bases and nick the damaged strand
- some of these proteins also remove the damaged region
DNA Pol I in DNA repair
-Can remove the damaged region (exo- or endonuclease)
-then fills in the previously damaged region
=-DNA ligase seals the final nick
HNPCC (hereditary nonpolposis colorectal cancer)
A defect in mismatch repair is responsible for this
One of the most common inherited cancers
DNA mutations
Can be causes by other mechanisms besides replication errors
Spontaneous mutations
Exposure to chemicals or radiation
-cigs
UV light
Causes pyrimidine dimers
-usually thymine dimers
UV light damaged DNA repair
UV specific endonuclease
-cuts DNA on both sides of damage and removes it, gap filled in by repair DNA polymerase (Pol I in prokaryotes)
Xeroderma pigmentosum
Results from deficiency in excision endonuclease
