topic 5 - DNA replication

Question 1

how often do Eukaryotic Chromosomes Replicate? what is a challenge of this?

Answer 1

• once per cell cycle
• multiple origins of replication

Question 2

What is a requirement of deoxynucleoside
triphosphate

Answer 2

primer:template junction

Question 3

what is processivity? Name an example of a processive enzyme and its function

Answer 3

an enzyme’s ability to catalyze “consecutive reactions without releasing its substrate”
- average number of nucleotides added each time when an
enzyme binds a primer:template junction

• DNA polymerase
• catalyzes DNA synthesis using a single active site for any dNTP

Question 4

functions of DNA polymerase

Answer 4

1. sterically distinguish between dNTPs (3’-OH) and its rNTPs (2’-OH + 3’-OH) precursors
2. show kinetic selectivity for adding correct base-paired dNTP (i.e., A-T and G-C)
3. initiate synthesis using either RNA or DNA primer annealed to the template

Question 5

describe the DNA polymerase palm analogy

Answer 5

finger - bind + enclose dNTP with
catalytic metal ions in palm

thumb - maintaining: correct position of primer and active site; and strong association b/ween DNA pol and its substrate

palm - catalytic site, monitor bp-ing of the most recently added nucleotides

Question 6

What removes RNA primers from RNA:DNA hybrid to complete DNA
synthesis?

Answer 6

RNase H

Question 7

replacement of DNA Pol α with
DNA Pol ε at the ______ strand
and Pol δ at the ______ strand

Answer 7

polymerase switching:
replacement of DNA Pol α with
DNA Pol ε at the leading strand
and Pol δ at the lagging strand

Question 8

what is a replicon

Answer 8

DNA replicated from a particular origin of replication

Question 9

how many proteins does the Origin Recognition Complex have and what is its function

Answer 9

• 6-protein complex
• ## recognizes replicators such as DNA helicase

Question 10

sequence of Replicator inactivation by DNA Replication

Answer 10

1. 3 and 5 activate
2. 2 and 4 activate by extension of 3 and 5
3. 1 turns on
4. they each become inactive after they’ve replicated to prevent repeats

Question 11

in what phase does origin activation occur? What does it trigger?

Answer 11

S phase
-triggers replicator associated
complex to initiate DNA unwinding and DNA Pol recruitment

Question 12

In what phase does replicator selection occur? what does this process do?

Answer 12

• G1 phase
• identify sequences that will direct initiation of replication

Question 13

describe the steps in helicase loading:
What stage of the cell cycle does this occur?

Answer 13

1. recognition of the replicator by the eukaryotic initiator, ORC
2. ORC recruits helicase loading proteins (Cdc6 and Cdt1) and Mcm2-7 helicase (costs ATP)

• during G1

Question 14

describe helicase activation:

Answer 14

• 2 kinases (CDK and DDK) phosphorylate Sld2/3 and Mcm2-7
• CMG complex forms (Cdc45/Mvm2-7/GINS)
• DNA Pol ε (leading strand) recruited BEFORE unwinding
• DNA Pol α/primase and DNA Pol
  δ recruited after unwinding

Question 15

impact of high vs low cdk

Answer 15

low CDK: loading of euk DNA helicase
high CDK: initiate DNA replication

Question 16

relative amounts of CDK in each stage of the cell cycle:

Answer 16

G1: CDK = low, helicase is loaded, BUT can’t be activated
S: CDK = high, helicase activated to initiate DNA replication
* used helicase is disassembled
after DNA replication
S, G2, and M: CDK stays high, prevents loading new helicase; restricts 1 DNA replication/cell cycle

Question 17

What recognizes the replicator?

Answer 17

initiator protein

Question 18

Which phase:
ORC + helicase loading proteins → assembly of DNA helicase on the replicator

Answer 18

G1 phase

Question 19

Which phase:
activation of helicase → DNA unwinding and assembly of DNA Pols to synthesize RNA primers

Answer 19

S phase

Question 20

circular DNA vs linear DNA replication

Answer 20

• circular DNA can be completely replicated
• separation of the two daughter DNA molecules by topo II

Question 21

why does a little bit of DNA get lost during replication?

Answer 21

since a primer is needed for replication, the end segment doesn’t have anything to use as its primer so that section doesn’t get replicated.

Question 22

solutions to prevent ends from not being replicated:

Answer 22

1. protein as primer instead of RNA at each end
2. use of telomere sequence

Question 23

How do proteins substitute primers at the ends of chromosomes?

Answer 23

• OH of an amino acid substitutes for 3’-OH of RNA primer
• protein allows priming at last nucleotide -> no sequence is lost

Question 24

how does the telomere sequence substitute primers?

Answer 24

• heat-to-tail repeats of a TG-rich DNA sequence
• serve as origin of replication for 3’
• use telomerase as the DNA pol
• e.g., human telomeres have head-to-tail repeats of 5’-TTAGGGTTAGGGTTAGGG-3’

Question 25

example of a ribonucleoprotein complex

Answer 25

telomerase

Question 26

components of ribonucleoprotein complex:

Answer 26

1. RNA subunit
2. reverse transcriptase

Question 27

compare Telomerase vs DNA Polymerase

Answer 27

telomerase -
* require template
* extend at 3’
* use the same nucleotide precursors
* act in processive manner
* telomerase has RNA component
* RNA:DNA helicase activity (displace RNA template from DNA for repeated rounds of synthesis)

polymerase -
* requires exogenous template
* use ssRNA substrate to make ssDNA (i.e., able to reverse transcribe)

Question 28

which piece of telomerase serves as the template?

Answer 28

RNA subunit

Question 29

how many nucleotides does telomerase add at a time?

Answer 29

6 nucleotide repeats

Question 30

Is DNA ss or ds after telomerase does its thing?

Answer 30

ss due to same issue of not being able to fill the gap of the primer (3’ overhang)

Question 31

what is shelterin and what are some of its functions?

Answer 31

set of proteins that bind to and protect telomere sequences at the end of euk chromosomes

• protect telomeres against chromosome joining
• regulates telomere length

Question 32

how does broken DNA signal a response?

Answer 32

broken DNA end (ATR pathway)
ss section (ATM pathway)