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MBIO2020 > 1.2 - Replication > Flashcards

1.2 - Replication Flashcards

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1
Q

what are the basics of replication in a prokaryote?

A

-bi directional
-has one initiation site (oriC in E.coli)
-has one termination site (ter in E.coli)

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2
Q

what are the features of oriC?

A

-AT rich region (easier to break because only a double hydrogen bond)
-binding sites for the replication initiation protein DnaA (ATP bound, active form)
-GATC methylation sites
-binding sites for FIS protein (inhibition of replication initiation) and IHF protein (DNA bending assistance)

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3
Q

what are the steps for the initiation of replication?

A

-the initial binding of ATP-DnaA to the higher affinity box allows cooperative binding of ATP-DnaA to the lower affinity boxes
-cooperative binding causes topological stress which results in the unwinding of the DNA in the AT-rich region (weaker bonds)
-single stranded DNA binding proteins stabilize the DNA and prevent helix formation (keeps it apart)
-HU protein assists with unwinding the AT-rich region
-for each strand 6 DnaC monomers will load 6 DnaB monomers (helicase)
-monomers of DnaB form a hexameric ring around each strand and the DnaC monomers dissociate after ATP-hydrolysis occurs because they are not needed
-DnaG (primase) is then loaded behind each helicase
-sliding clamp is loaded to be used by DNA polymerase III
-the hydrolysis of ATP-DnaA to ADP-DnaA promotes dissociation of the DnaA
-primase puts down RNA primers

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4
Q

how does the affinity for ATP-DnaA work?

A

-longer sequence = higher
-single stranded boxes have higher affinity than double strand boxes

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5
Q

why does helicase need to be loaded?

A

-it has trouble binding with the single stranded DNA binding proteins present

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6
Q

what does DNA polymerase III recognize on the RNA primers?

A

-the 3’ OH ends

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7
Q

what is the sliding clamp a subunit of?

A

-beta subunit of DNA polymerase III

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8
Q

what causes the 5’-3’ directionality?

A

-orientation of the sugar molecules in the sugar-phosphate backbone

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9
Q

what stabilizes the structure of DNA?

A

-the bases
-bases stack and have hydrogen bonds (2 modes of stabilization)

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10
Q

what is the precursor of each new nucleotide in the DNA strand?

A

-deoxynucleoside 5’ triphosphate (dNTP, N=A,T,C,G)
-the exact version will be determined by the template strand sequence

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11
Q

what must be present in order to DNA synthesis to occur?

A

-free OH group at the 3’ end of the molecule (sugar)

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12
Q

what happens to the deoxynucleoside triphosphate in the insertion process?

A

-the two terminal phosphates are removed
-the remaining phosphate is bonded to a deoxyribose in the growing chain (3’ OH)

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13
Q

what is PP subscript i? where does it come from?

A

-pyrophosphate
-high energy phosphates
-comes from the cleaves of terminal phosphates after the insertion process

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14
Q

what is the directionality of DNA synthesis?

A

-always proceeds from the 5’ - 3’ end (how DNA polymerase works)
-5’ phosphate of the incoming molecule is attached to the 3’ hydroxyl of the previously added molecule
-read 3’ - 5’
-replicate 5’ - 3’

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15
Q

what is the role of the DNA polymerases?

A

-DNA polymerase III reads the template and incorporates new nucleotides as per base pairing rules
-DNA polymerase I removes the RNA primer and fills in the gaps

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16
Q

why cant DNA polymerase start synthesis on its own?

A

-can only add onto pre-existing 3’ OH groups
-therefore it needs a nucleic acid molecule to start (primer of 11-12 nucleotides)

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17
Q

what fuels helicase activity?

A

-energy release from ATP hydrolysis

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18
Q

what does helicase do?

A

-opens up the strands just ahead of the replication fork

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19
Q

what happens as helicase unwinds the DNA?

A

-positive supercoils form and eventually is brought to a point where no more opening can happen (overwhelmed DNA, under torsion)

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20
Q

what relieves the positive supercoils from the DNA strands?

A

-DNA gyrase (special type II topoisomerase)
-inserts negative supercoils into the DNA which cancels out the positive ones
-inserts them by making 2 nicks in the ssDNA

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21
Q

what is the role of topoisomerases?

A

-mediate DNA supercoiling (insert or remove them)

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22
Q

what is negative supercoiling?

A

-when DNA is twisted about its axis in the opposite sense from the right handed double helix
-found in most cells

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23
Q

what is positive supercoiling?

A

-results when DNA is twisted in the same direction

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24
Q

what is the overall process of leading strand synthesis?

A

-strand is already oriented in the 3’ - 5’ direction to be read (DNA polymerase works towards the fork joint away from oriC to synthesize 5’ - 3’)
-strand grows continuously because the 3’ OH is always free
-only primed once

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25
what is the overall process of the lagging strand synthesis?
-strand is oriented in the 5' - 3' direction (fork joint = 3' end, oriC = 5' end) (DNA polymerase works towards oriC and away from the fork joint to synthesize 5' - 3') -strand grows discontinuously since it lacks free 3' OH groups (on the opposite end) -made in okazaki fragments and joined later to form a continuous strand -primed multiple times
26
what is the steps to lagging strand synthesis?
-primase places an RNA primer toward oriC away from the fork joint -DNA polymerase III synthesizes DNA toward oriC -synthesis stops at the origin forming the 1st okazaki fragment -replication fork opens more and another primer is placed behind the first fragment to begin the 2nd fragment -synthesis of the 2nd fragment stops when DNA polymerase III runs into the primer for the 1st fragment -leaves a small gap when it approaches the primer -process repeats
27
how do the gaps in the lagging strand get fixed?
-DNA polymerase I recognizes the gap and closes it by removing the RNA primer and synthesizing DNA in its place -DNA ligase (ligA + ligB) completes the last phosphodiester bond between the 3' OH and the 5' PO4^2- using ATP for energy
28
what type of activity does DNA polymerase I have that allows it to remove the RNA primers?
-5' - 3' exonuclease activity
29
why must DNA ligase complete the last bond?
-DNA polymerase can only extend from the 3' OH -it cannot catalyze a phosphodiester linkage with a 5' end of a pre existing nucleotide
30
what is the replisome?
-large complex of replication proteins that come together to mediate DNA replication -DNA polymerase III (mediates polymerization) -DNA gyrase (removes positive supercoils) -SSBs (prevent helix reformation) -helicase (unwinds the DNA) -primase (primes the DNA) -tau protein (holds the 2 polymerases together) -sliding clamp
31
what does helicase and primase form?
-the primosome -subcomplex of the replisome
32
what can be said about the movement of DNA replication?
-DNA moves through the DNA polymerase III rather than it moving along the DNA
33
what is abnormal about the movement of DNA replication at the lagging strand in terms of DNA polymerase III?
-when the lagging strand nears completion of a fragment primase will make a new primer and the primosome will load a new sliding beta-clamp -once the fragment is complete, DNA polymerase will dissociate from the old clamp and associate with the new clamp -it then begins synthesis again
34
what is the process of termination in replication?
-replication forks meet up at the ter site -the tus protein will bind to the ter site and stop the replication forks -chromosomes may be interlocked (catenanes) -topoisomerase IV (type II topoisomerase) cuts both strands on one chromosome (ds break) and passes the intact chromosome through the break and then rejoins strands -process is called decatenation
35
how does the incorporation of dNTP by DNA polymerase III and I reduce error rate?
-because of complementary base pairing rules
36
how does DNA polymerase I's exonuclease activity to remove RNA primers reduce error rate? why is it important for lagging strand sythesis?
-RNA primers can have a mismatch, but this mistake is only temporary as it gets removed by the DNA polymerase I -this is very important for the lagging strand because multiple primers get placed
37
in what direction does DNA polymerase I have exonuclease activity?
- 5' - 3' and 3' - 5' (both directions)
38
in what direction does DNA polymerase III have exonuclease activity?
- 3' - 5'
39
how does the 3' - 5' exonuclease activity of both DNA polymerase I and III reduce error rate and how do they achieve this?
-allows them to back up and remove any mis-basepaired nucleotides -DNA polymerase III has a DnaQ proofreading subunit -DNA polymerase I has dual function of polymerization and proofreading -can sense mismatched basepairs by distortion in the helix
40
what is another way of reducing error rate?
-methyl - directed mismatch repair
41
how do we name genes and how do we name proteins?
-gene names are italicized and lowered cases for dna (ex: dnaA) -protein names are not italicized and are capitalized for Dna (ex DnaA)
42
why must replication be tightly controlled?
-needs to coincide with cell division -therefore the initiation of replication needs to be tightly controlled
43
how is replication initiation controlled?
-by DnaA (replication initiation protein) (has multiple regulatory mechanisms to block or inactivate DnaA-ATP (its active form)) -methylation status
44
how does methylation status control replication initiation?
-GATC sequences are interspearsed through chromosomal DNA with a high concentration in oriC -in a non-replicative state, the adenine in the sequence is methylated (CH3 added) which is done by DNA adenine methylase (DAM) -when DNA is newly replicated, the chromosomes will be hemi-methylated for about 10 minutes before DAM comes in to fully methylate them
45
what is the control mechanism of replication initiation that is competition for oriC binding?
-once replication is initiated, newly synthesized oriC sequences are hemi-methylated -SeqA (sequestration protein A) will bind and block the DnaA binding sites to prevent re-firing of oriC
46
what is the control mechanisms of replication initiation that represses dnaA expression?
-the dnaA gene is located close to the oriC region -expression of the dnaA gene is autoregulated as DnaA-ATP will bind to DnaA boxes within its own promotor region -this only happens in a fully methylated state which means that the dnaA gene espression is also subject to SeqA control as it covers the binding sites for DnaA-ATP (only when hemi-methylated) -overall, if there is lots of DnaA-ATP, it will bind to the box in its own promotor region and block transcription from happening
47
what is the goal of dnaA expression?
-expression is activated to initiate replication -after replication the expression is repressed
48
what is the control mechanism of replication initiation that is reducing DnaA-ATP levels?
-binding of DnaA-ATP can be prevented within the oriC region by decreasing the ratio of DnaA-ATP to DnaA-ADP -this is controlled by ATPase HdaA which associates with DNA in the proximity of the replisome -promotes hydrolysis of DnaA-ATP to DnaA-ADP in a replication dependent manner (as replication happens) -also results as a combination of mechanisms (reaches a max amount when replication initiation first happens, and then weaning off after)
49
how does the replication rate compare to the generation time within E.coli?
-genome replication takes about 40 minutes -in low nutrient conditions, generation time is 60 minutes -in high nutrient conditions, generation time is 20 minutes
50
how do E.coli get around the short generation time in comparison to the replication rate?
-a new replication round is initiated before the previous round is complete -this means that genes close to oriC will be present in more than 1 copy (ex: dnaA) -newly synthesized DNA is methylated (SeqA loses control) and DnaA-ATP can bind to initiate another replication round
51
what is the associated process with the topoisomerase IV separating intertwined DNA?
-MukBEF complex -a protein complex that binds to the nucleoid and localizes to distinct regions in the cell to assist with the separation of replicated chromosomes (oriC, Ter, mid point) -together form the structural maintenance of chromosome complex (SMC complex)
52
how is the divisome formed?
-starts with the attachment of FtsZ molecules in a ring formation around the center of the cell (cell division plane) -forms after the DNA has replicated as it needs to be placed inbetween the duplicated nucleoids -the Min system (MinC, MinD, Min) helps to guide the formation of the FtsZ ring to the midpoint of the cell
53
how does the Min System work?
-MinCD forms a spiral structure on the inner surface of the cytoplasmic membrane along the long axis of the cell (role = to prevent FtsZ ring formation from inhibiting cell division) -MinE moves from pole to pole pushing MinC and MinE aside -center of the cell will have the lowest concentration of Min proteins to allow for FtsZ formation
54
what does the formation of the FtsZ ring attract?
-ATPase FtsA -ZipA
55
where does the energy for the polymerization and depolymerization of the FtsZ ring come from?
-GTP
56
what is the role of ZipA?
-anchors the FtsZ ring to the cytoplasmic membrane to stabilize it
57
what is the role of ATPase FtsA?
-recruits FtsZ and other divisome proteins (more FtsA, FtsI, and FtsK) -helps to connect the ring to the cytoplasmic membrane
58
what is the role of FtsI?
-peptidoglycan synthesis
59
what is role of FtsK?
-chromosome seperation
60
what does the divisome direct synthesis of?
-a new cytoplasmic membrane and cell wall material (forms the division septum
61
what happens as the cell constricts?what does this then trigger?
-the FtsZ ring begins to deploymerize -triggers the inward growth of wall materials to form the septum and seal off one daughter cell from the other (complete seperation)
62
how do FtsK and topoisomerase IV interact to separate chromosomes?
-FtsK moves chromosomes (translocates) at the division septum -Ftsk also regulates decatenation of chromosomes by interacting with topoisomerase IV -topoisomerase IV interacts with MukB (chromosome condensation protein) to remove catenanes before the DNA is condensed and packaged into the cell

MBIO2020 (12 decks)

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