Study Questions Set 4

Question 1

List proteins that are involved in DNA replication in E. coli. remember, this is prokaryotic.

Answer 1

DnaA
Helicase (DnaB)
DnaC
SSB
Topoisomerase II
Primase (DnaG)
DNA polymerase III
RNase H
DNApol I
DNA ligase

Question 2

Tell me about DnaA

Answer 2

prokaryotic. the protein that initiates replication at the OriC.it will recognise 4 DNA boxes (9bp, AT rich) and AT rich 13-bp sequences. 10-20 DnaA proteins and 4 DnaA boxes form an initial complex at the 9bp, no ATP required, and then binding and opening a complex at the 13bp region while using ATP. it will initiate replication only if the DNA is supercoiled

Question 3

Tell me about DnaB

Answer 3

prokaryotic. helicase/DnaB is going to travel down the dsDNA and separate the strands, using ATP along the way. it will move towards the duplex region of the replication fork and it will remain processive - it will only stop until it is either removed by proteins, or it finishes unzipping the whole molecule

Question 4

tell me about DnaC

Answer 4

prokaryotic. it will escort DnaB to be escorted to form the pre-priming complex.

Question 5

tell me about SSB

Answer 5

single stranded binding protein. it is a protein that binds to ssDNA and prevents it from re-associating. it undergoes cooperative binding (the binding of one molecule leads to the binding of another molecule). it prevents pairing of the DNA into dsDNA

Question 6

tell me about topoisomerase II (gyrase)

Answer 6

prokaryotic. it will convert the positive supercoils forming (because of DnaB) into negative supercoils

Question 7

tell me about DnaG (primase)

Answer 7

prokaryotic. because DNApol can only extend on existing strands, RNApol forms a primer for the DNApol to bind to (because remember DNApol can only bind to 3’OH). well, DnaG will function with helicase (DnaB) to form the next priming site (primosome)

Question 8

DNApol III

Answer 8

binding of DNApol III means initiation is over and polymerization starts, it will synthesize a new DNA strand using the old one as a template

Question 9

RNase H

Answer 9

degrades RNA primer

Question 10

DNApol I

Answer 10

exonuclease and endonuclease activity, involved in fixing of errors during synthesis (3’–>5’) and removal of primers (5’–>3’). it will fill the gaps of the RNA primers with dNTPs

Question 11

DNA ligase

Answer 11

ligate 2 fragments where there is gap, important for lagging strand where okazaki fragments are formed

Question 12

What is meant by replication being bidirectional?

Answer 12

from the origin, DNA replication occurs in both directions via the replication fork, the replication, and leading and lagging strands

Question 13

what is meant by replication being semiconservative

Answer 13

DNA replication involves the serving of the leading and lagging strand as templates, so the new dsDNA molecule has 1 old and 1 new strand

Question 14

what is meant by replication being continuous?

Answer 14

leading strand replication, because it requires only one primer

Question 15

what is meant by replication beind discontinuous

Answer 15

lagging strand replication, because it involves many primers and okazaki fragments. happens because the DNApol works 5’–>3’

Question 16

Contrast the role of DNApol I and III during prokaryotic replication

Answer 16

III polymerizes new DNA on the RNA primer using the template strand. I will remove the RNA primers and replace them with dNTPs, and also fix any errors made by III

Question 17

Which subunit of DNApol III provides processivity? which protein loads this subunit on the DNA?

Answer 17

The B-subunit functions as a clamp, but associates with the core protein at the end of the growing strand, thereby increasing the processivity of the strand. the gamma subunit will load the beta subunit onto the DNA with the use of ATP

Question 18

how can the discontinuous synthesis of the lagging strand keep up with the continuous synthesis of the leading strand?

Answer 18

DNApol III allows for the leading and lagging strand to be replicated at the same pace at the same time
trombone model has the lagging strand stay linear in the region that DNApol III binds to

Question 19

why is decatenation required in circular DNAs?

Answer 19

the loops of DNA are interlocked at the end of replication due to the formation of the theta structure during replication. topo IV is responsible for nicking and removal of the replicated dsDNA

Question 20

why do eukaryotes need telomeres but prokaryotes do not?

Answer 20

eukaryotes lose the ends of chromosomes at the end of replication because they have nothing to join the ends to. the lagging strand is unable to fill the final RNA fragment that is removed (no 3’OH forDNApol to build on), therefore approx 100bp are lost from 5’ ends with each mitosis. telomeres make sure that only repetitive, unecessary genes are lost. prokaryotes are circular and they will just join the 2 ends of the DNA. telomeres are GC rich, like kinetochores. telomeres form a t-loop at the end.

Question 21

what would we need in vitro to make DNA?

Answer 21

DNApol I, primase, DnaA, DnaB, DnaC, SSB, topoisomerase II, DNA ligase

Question 22

What properties would you expect an E. coli cell to have if it had a temperature – sensitive mutation in the gene for DNA polymerase I?

Answer 22

At appropriate temperature, the inability to remove the last ribonucleotide or fill the gaps from RNA primers; likely not viable, but DNA polymerase III might accommodate for filling in

Question 23

Compare and contrast major euk and pro DNApols

Answer 23

Prokaryotic: DNA polymerase I (removes RNA primers and fills in necessary nucleotides; not highly processive; exonuclease activity)
Prokaryotic: DNA polymerase III (main replication polymerase; highly processive; exonuclease activity)
Eukaryotic: 9 different DNA polymerases
α – unusual; some primase function, initiation of replication. makes a short piece of DNA because there’s no proofreading ability. elongation via DNApol activity.
δ – takes over from alpha and elongates lagging strand; processive; elongation of okazaki DNA fragments
ε – possibly involved in elongation of leading strand, takes over from Pol a. continues leading strand synthesis.
MCM = helicase
cdc6/cdt1 = helciase loader
FEN1 = endo/exonuclease, removes primer, sometimes needs RnaH1

Question 24

What is telomerase and why is it important?

Answer 24

telomerase: the enzyme that is responsible for the creation of telomerase. telomerase has an RNA template that it carries around, it complimentary to the telomerase repeat. it is a ribonuceloprotein - protein RNA activity. it also has reverse transcriptase activity, in that it makes RNA from DNA. after extension of the 3’OH overhang of the leading strand, using its own RNA template, a new RNA primer for the lagging strand is made by RNA primase, and then extended by DNApol. this terminal RNA primer on the C-rich strand will be degraded, resulting in another 3’ overhang of the G-rich leading strand.. and then there is the formation of that t-loop.

Question 25

What is the major difference between bacterial and eukaryotic replication that allows a eukaryotic cell to replicate its DNA in a reasonable amount of time?

Answer 25

eukaryotes have multiples sites of replication, while prok have only 1. therefore, euk will go faster.

Question 26

Describe the events that occur at an origin of replication during initiation of replication in E. coli?

Answer 26

• origin of replication (spreads over the area of ~245 bp)  4 repetitive DnaA boxes and 3 A-T rich 13 bp sequences) is recognized by specific proteins  DnaA recognizes the DnaA boxes  10-20 DnaA proteins and 4 DnaA boxes form an initial complex  initiates replication only if DNA is negatively supercoiled  binding and opening occur at the 13 bp sequences and require ATP

Question 27

What are cis- elements? What are trans- factors?

Answer 27

• cis elements: specific nucleotide sequences with a precise distribution of acceptors and donors that regulate the expression of genes located on the same strand
• trans factors: trans-acting functions; diffuse through the cell/nucleus  proteins that can recognize and act on cis elements to regulate transcription

Question 28

17 Is the following statement true or false: Regardless of whether a gene is expressed in a given cell type, it will replicate at the same, characteristic time during S phase. Explain your reasoning.

Answer 28

• False; clusters of euchromatin replicons replicate first (less condensed)
• Multiple replicons (50,000 – 100,000) that are 40-100 kb long
• Clusters of 20-50 replicons initiate simultaneously (euchromatin first)

Question 29

Thinking question: Is making of RNA primers (by primase), which have to be subsequently removed and replaced with dNTPs (by DNA polymerase I) actually wasteful and energetically inefficient process? (Hint: think about fidelity of primer-making vs. proofreading capability of DNA pol I, in other words what is the only purpose of RNA primers – is the accuracy of this process very important at this point?)

Answer 29

• RNA primers need not be accurate since they will be excised and replaced by DNA polymerase; more efficient than using DNA primers which would need to be exact
• Since a 3’ OH group is needed, these primers are necessary to achieve replication and since DNA pol I has proofreading capability, this process is not overly wasteful

Question 30

• RNA primers need not be accurate since they will be excised and replaced by DNA polymerase; more efficient than using DNA primers which would need to be exact

Answer 30

• Since the ends of linear chromosomes are slowly degraded over time (approx. 100 bp each mitosis), this area of re-association (highly repetitive bases) is reduced and becomes less and less effective for re-annealing