02/06/2025 Flashcards
what are the general steps to DNA replication
initiation, elongation, termination
what are the requirements for initiation of DNA replication in bacteria?
1.) the GATC sites must be fully methylated at both sides of the strands in ORDER for the AT rich regions to be opened WHEN the DnaA boxes are fully occupied by DnaA binding proteis
what are DnaA binding proteins
they are proteins that recognize the sequence of the DnaA boxes which are recognition sequences, with assistance, these can open the AT rich regions
What is the role of helicase? what does it NOT do?
disrupts DNA helix by breaking H-bonds between the strands, it does NOT create the fork
As helicase opens the fork more, what is formed ahead of the fork? What must be used to counteract this effect?
positive supercoils form ahead of the fork
Gyrase/ topoisomerase II acts to relieve this tension by inducing negative supercoils
What are single-stranded binding proteins
Proteins that bind to separated DNA strands to keep them apart
What does it mean when something is DNA dependent
it means that it requires DNA as a template
what does it mean when something is RNA dependent
it uses RNA as a template
what is a DNA dependent DNA polymerase
uses DNA to synthesize DNA
what is a DNA dependent RNA polymerase
uses DNA to synthesize RNA
what is a RNA dependent DNA polymerase
it uses RNA to synthesize DNA
what is an RNA dependent RNA polymerase
it uses RNA to synthesize RNA
what is the role of primase and what would happen if we didn’t have it?
primase adds an RNA primer to the template strand which provides a 3’ OH that DNA pol III can synthesize onto
before DNA pol III can synthesize new DNA, what must happen first?
primase must make an RNA primer to provide a 3’ OH group
what is the leading strand?
the leading strand is continuously synthesized by DNA pol III towards the replication fork
how many primers does the leading strand require?
it requires 1 primer
what is the lagging strand
the lagging strand is synthesized in spurts by DNA pol III as it is synthesized away from the fork
how is the lagging strand synthesized as the fork opens?
as the fork opens, another primer is added by primase.
when DNA pol III is done synthesizing one fragment, it loops back on itself and begins to synthesize another fragment on the new primer
how many primers does the lagging strand require
multiple
what is an okazaki fragment
they are the fragments that make up the lagging stand
when two okazaki fragments run into eachother, how are they fused?
DNA pol I acts to replace the RNA with DNA but Ligase forms the final bond between the replaced nucleotides and the DNA strand synthesized by DNA III
what is the role of DNA pol I
it replaces the RNA primer with DNA
what is the role of ligase
ligase forms the final phosphodiester bond between the 3’OH and 5’ phosphate group on the replaced okazaki fragments
without ligase, would DNA pol 1 be able to fuse the okazaki fragments
no, ligase is required to form the final connection
what is the ter protein’s role
it binds to the termination sequence and prevents advancement of the replication fork
what is the DNA pol III holoenzyme
it describes the 10 subunit complex that makes up DNA pol III
what is the overall structural composition of DNA pol III
it is a quaternary structure as it interacts with other polypeptides to form its 10 subunits
what is the overall structural composition of DNA pol I
it is a tertiary structure as it is composed of a single polypeptide and does not have other subunits
describe the levels of polypeptide structure
primary is the strand itself and sequence of amino acids
secondary is local folding
tertiary is the 3D structure
quaternary is when the 3D structure interacts with other polypeptides
why does a gene code for a polypeptide and not a protein
a polypeptide is a sequence of amino acids while a protein describes its function and may require various subunits to do so
what does the alpha subunit in the DNA pol III do
it synthesizes DNA at the back of the polymerase
what does the ε subunit do in DNA pol III
it regulates the proofreading function
what is the proofreading function of DNA pol III
an enzyme in DNA pol III can read in the 3’-5’ direction and remove mismatched base pairs
what is the 3’ exonuclease site
it is where the proofreading occurs
when a base pair is mismatched, the end of the new strand will move to the 3’ exonuclease site where it will be digested in the 3’-5’ direction and then resynthesized in the 5’ to 3’ direction
what direction is DNA digested in the 3’ exonuclease site
3’-5’
what is the difference between exonucleases and endonucleases
exonucleases cleave DNA/RNA at their ends
endonucleases cleave DNA/RNA internally
what does proofreading DNA pol III or DNA pol I
DNA pol III
when DNA pol I is replacing the RNA primer, which way is its exonuclease activity and which way is its polymerase activity? How does it digest and form?
it removes the primer and synthesizes the new DNA in the 5’ to 3’ direction
when does DNA pol III stop?
when it hits the primer from the next fragment, it cannot seal itself
During DNA replication, the leading and lagging strands are synthesized simultaneously by a single DNA polymerase III holoenzyme. Since DNA polymerase can only synthesize DNA in the 5’ → 3’ direction, how does the lagging strand, which is oriented in the opposite direction, get replicated efficiently?
the lagging strand gets looped out so the DNA polymerase can stay near the newly formed primers and easily “jump” over to start synthesizing at a new RNA primer
what does the function of looping out allow?
it allows coordination between the leading and lagging strand and allows it to be synthesized discontinuously
what is the specific function of DNA ligase, what type of bond does it form
DNA ligases catalyze a phosphodiester bond and connect DNA fragments
between which two groups is the phosphodiester bond formed between in DNA pol?
the innermost phosphate group of the incoming nucleoside triphosphate
the 3’OH of the sugar of the previous deoxynucleotides
when the nucleoside triphosphate and 3’OH form a phosphodiester bond, what is formed as a byproduct of the triphosphate
the last two phosphate groups in a pyrophosphate form (PPi)
what is the exonuclease function of DNA pol I
it is the digestion of RNA in the 5’-3’ direction
what is the polymerase function of DNA pol I
it is the formation of DNA in the 5’-3’ direction
what is the replisome
it is the coordination complex of all the enzymes working in sequence and it is associated with two DNA polymerase holoenzymes forming the leading and lagging strand
in order for the replisome to coordinate properly, what must be done to the lagging strand
it must loop out
what is a dimeric DNA polymerase?
which way does it move
two DNA pol III proteins act in concert to replicate the leading and lagging strands
it moves as a unit towards the replication forks
what is opposite to the origin of replication and why is this significant
at the opposite of the oriC, there are termination sequences where termination binding proteins can adhere to and stop further replication or opening of the fork
At the end of bacterial replication, what often results
intertwined molecules called catenanes
how are catenanes separated?
by action of topoisomerases
Why is DNA pol III not 100% efficient
molecules are dynamic
why does DNA replication have a high rate of fidelity
1) instability of mismatched base pairs
2) configuration of DNA pol active site
3) proofreading function of DNA pol III
why does the instability of mismatched base pairs contribute to the fidelity of DNA rep
due to complementarity, complementary base pairs are more stable than mismatched ones
this accounts for errors 1 per 1000 nucleotides
why does the configuration of the DNA pol III active site mater in DNA replication
it helps prevent errors (rate: 1 in 1 million) due to the induced fit phenomenon that occurs in enzymes. It is unlikely catalyze bond formation between mismatched base pairs
why is the proofreading function of DNA pol III important
it significantly reduces the error in DNA replication as it can identify mismatched pairs, move backwards in a 3’-5’ direction and remove the incorrect base pairs in its 3’ exonuclease site before returning to regular replication
how are bacterial cells able to divide at such a fast rate given the amount of time it takes for DNA to replicate
they have an ability to control the initiation of replication at the OriC and can fire off multiple rounds of replication before the first one is even done
why can bacterial cells not immediately fire off the origin of replication
it takes time for methylase to completely methylate the GATC regions and it takes time to synthesize a sufficient amount of DnaA protein for binding
when DNA replication begins, what happens to the fully methylated sites?
they are hemi-methylated and cannot start replication again until they are fully methylated
when DNA replication begins, what happens to DnaA binding proteins?
there is an insufficient amount of them available to bind to the DnaA binding sites, so it takes time to produce more.
