Lecture 10 Flashcards
What model does DNA replication follow
Semi-conservative model
The substrates for
DNA synthesis are?
Nucleoside triphosphates (NTPs)
When each nucleotide is incorporated into the growing DNA strand, what bond forms with what and what bond breaks?
When each nucleotide is incorporated into the growing DNA strand, it forms a new phosphodiester bond with the last nucleotide added, and the bond between the first phosphate, Pa, and the second phosphate, Pb, of the NTP breaks.
What is DNA replication
duplication of chromosomes for the purpose of cell division
Replication occurs by addition of NMPs to the ___ of a nascent (new) strand
3’-OH
The correct NTP substrate is selected based on
its base pair complementarity with the nucleotides in the existing strand to be duplicated. This strand acts as a “template”, dictating the nucleotide sequence of the newly-synthesized “daughter” strand
Where do incoming nucleotides get positioned?
incoming nucleotide gets positioned adjacent to the 1st non-paired nucleotide on the template strand through Watson-Crick base pairing between the incoming dNTP base and the template base
Where is the nucleophilic attack done on during DNA replication and by which molecule
nucleophilic attack by the 3’-OH of the most recently added nucleotide (on the primer/daughter strand) on the alpha-phosphate of the incoming dNTP
Which way does DNA synthesis occur?
DNA synthesis always occurs in the 5’’ -> 3’ direction, with dNMPs being added iteratively to the 3’ end of the primer/daughter strand
Why does DNA synthesis occur 5’ -> 3’
because of the requirement for the 3’- OH from the primer strand and the 5’ phosphate on the incoming dNTP substrate
The raw materials for DNA replication are (5)
- a DNA template
- deoxynucleoside triphosphates (dNTPs)
- a protein complex that includes the enzyme DNA polymerase (DNA is a polymer of nucleotides)
- a DNA or RNA ‘primer’ to provide a 3’ OH
- Mg2+ ions (cofactor for the polymerase)
How do you unwind DNA to expose the template strand bases?
In vitro this is done by heating up the DNA to form two separate template strands. Short DNA oligonucleotides (primers) are added that are complementary to a segment of the template strands. The mixture is cooled and the primers anneal to the template strand. DNA synthesis extends the primer strands in the 5’ ® 3’ direction
the DNA ends are labeled
according to the exposed end of the sugar:phosphate backbone.
DNA synthesis in vivo requires an ___ primer synthesized by an ___
polymerase
- RNA primer
- RNA polymerase
What are helicases
Enzymes that unwind DNA in vivo to expose the bases on each of the template strands
DNA replication is primed by?
a short RNA primer
RNA primer is synthesized by?
Primase, an RNA polymerase
DNA polymerases require a pre-existing what
DNA or RNA primer and template
How is the RNA primer removed after
at a later stage of replication and replaced by DNA
Difference between DNA and RNA polymerase?
DNA polymerases require a pre-existing DNA or RNA primer and template. They cannot synthesize DNA de novo. They must add incoming dNMPs to an existing primer with a 3’-OH. (RNA polymerases do not need a primer and can synthesize RNA from scratch.)
Where does DNA synthesis occur?
DNA synthesis occurs at replication forks simultaneously for both parent strands, always in the 5’ -> 3’ direction
Which strand is synthesized continuously?
Leading strand
Which strand is synthesized in short pieces?
Lagging strand
What are Okazaki fragments?
short pieces synthesized for the lagging strand
Both new strands are synthesized in a coordinated
fashion by
a single dimeric DNA polymerase III complex (DNA pol III)
How are the leading and lagging strands, which run in
opposite directions, both produced in the same direction by the DNA polymerase III complex?
The template for the lagging strand loops
What is the origins of replication
In all cells (eukaryotic, prokaryotic) with circular or linear DNA molecules, DNA replication begins at defined sequences termed “origins of replication” (ori).
Initiation of replication is controlled by?
proteins that recognize and bind to the DNA sequence of the origin of replication
Are there usually one or more origins
Often there are multiple origins
What has circular chromosomes with a single origin of replication - “Ori C”
Prokaryoates like E. coli
the ori opens up (DNA strands
are separated by a helicase) to form ___ replication forks
2 replication forks
replication is ____, with a DNA polymerase complex operating at each of the 2 forks
bidirectional
Prokaryotes vs eukaryotes in terms of ori
Prokaryotes have a single origin of replication, eukaryotic genomes
have thousands
Why isn’t the human genome replicated from a single origin
Replication of the human genome from a single origin with two forks on each chromosome (i.e. bidirectional replication)
would take weeks - need many origins of replication.
What are DNA polymerases
enzymes that catalyze polynucleotide chain elongation (nucleotide polymerization)
What is the role of DNA pol I
“proof-reading” and “nick translation” (connecting Okazaki fragments)
aka “clean-up polymerase”
replaces the RNA primer nucleotides (ribonucleotides) with deoxynucleotides in a
process called nick translation
proof-reading functions
What is role of DNA pol III
major polymerase involved in chain elongation during replication (also has proof-reading function)
main elongation enzyme
highly processive: adds thousands of nucleotides in 5’-3’ direction before falling off the DNA
What is an exonuclease (exo)
an enzyme that catalyses the hydrolysis of phosphodiester bonds at the end of nucleic acid molecules
What direction do exonucleases act ing
Some exonucleases act in the 5’ 3’direction, others in the 3’ 5’ direction
What direction does DNA pol I act in
DNA pol I has both 5’ 3’and 3’ 5’exonuclease activity
What direction does DNA pol III act in
DNA pol III only has 3’ 5’exonuclease activity
What is primase
Primase and helicase complex
What is the function of primase
Generates RNA primer
What is the fxn of helicase
Unwinds DNA
Fxn of SSBs?
Keep DNA unwound
Which DNA pol is the “clean up polymerase”
DNA pol I
Fxn of topoisomerase
relieves topological stress generated from unwinding by helicase
How many domains do DNA poly I have
3
What is nick translation?
When DNA pol I Removes the RNA primer one ribonucleotide at a time: 5’ 3’ exonuclease activity
Adds deoxyribonucleotides to the 3’ end of the adjacent Okazaki fragment (lagging strand): 5’ -> 3’ polymerase activity (polymerization always occurs 5’ -> 3’)
What is proof reading
Excises mismatched or defective nucleotides in the 3’ end of the Okazaki fragments: 3’ -> 5’ exonuclease
Does each okazaki fragment has its own RNA primer?
Yes
How are okazaki fragments removed
DNA pol I simultaneously polymerizes (extends) one end of an Okazaki fragment and excises the RNA primer of an adjacent Okazaki fragment one nucleotide at at time
What adds the dNMPs?
DNA pol I
What happens when the dNMPs are incorrect?
DNA pol I excises them (3’ 5’ exonuclease activity) and replaces them with the correct nucleotide.
Which side are the dNMPs added on the Okazaki fragments?
the 3’ end of one Okazaki fragment
Which side are ribonucleotides removed from?
5’ end of the adjacent RNA primer
The nick between two strands get translated along the strand in which direction?
5’->3’
The nick is sealed by what
DNA ligase
How many incorrect base pairs are there
1 for every 10,000 correct insertions
The specificity of replication is dictated by?
hydrogen bonding and shape complementarity between the bases on the template strand and the incoming bases as well as by correct fitting of the base pairs in the polymerase active site
How can DNA polymerase replicate DNA more faithfully than H bonding interactions between the bases alone can account for
some amino acid side chains of the polymerase active site form H bonds with the base pair: “base selection
What acts like molecular rulers?
H-bonds from bases in the minor groove to amino acid side chains in the active site of polymerase
What happens when C tries to pair with A, and G tries to pair with A
, if a C tries to pair with A, one of the bases would need to tilt to optimize H-bonding, which would then displace H-bond acceptors in the minor groove. If a G tries to pair with A the geometry would be even worse as both G and A are bicyclic (double-ringed) and would disrupt the spacing between the DNA backbones. Incorrect base pairing would exclude the pair from the active site of the polymerase, allowing it to migrate to the 3’ 5’ exonuclease site where the mismatched base is excised.
Phosphodiester bond is either not formed or formed but subsequently excised
What is fidelity
High accuracy
How many subunits is DNA pol consisted of
10 types
Which subunits form a clamp on each core polymerase complex (DNA pol III)?
A pair of B-subunits
Function of the B-B-clamps?
- each B-clamps encircles a template/daughter DNA complex like a clamp
- the B-clamp slides along the DNA and prevents dissociation of the polymerase - dramatically increases the processivity of the polymerase
What is processivity
the ability of an enzyme to repetitively continue its catalytic function without dissociating from its substrate.
