Module 02 - Section 02

Question 1

What is DNA replication?

Answer 1

Synthesis of 2 daughter DNA molecules identical to the parental DNA

Question 2

Requirements for DNA replications?

Answer 2

• All 4 nucleotides
• Intact DNA template
• Mg++ cofactor

Question 3

What are the 6 general principles of DNA replication?

Answer 3

(1) Semiconservative
(2) Initiated at specific site
(3) Typically bidirectional
(4) Semidiscontinuous
(5) RNA primers are needed to start
(6) Nucleases, polymerases and ligases replace the RNA primers with DNA and seal remaining nick

Question 4

What does semiconservative replication mean?

Answer 4

Each New DNA molecule conserves on strand from the parental DNA and the other strand is new

Question 5

What were the first 2 hypothese on the conservativeness of DNA replication?

Answer 5

(1) it was conservative, old DNA is made from parental DNA and daughter DNA from replication
(2) Parental stand and new daughter strand will be randomly mixed together creating strands having a patchwork of old and new DNA

Question 6

Where does replication start?

Answer 6

At specific DNA sequence called the origin of replication with the participation of an origin recognition system

Question 7

What is the replication fork?

Answer 7

The point where the parental duplex separates and the daughter duplexes form

Question 8

What is the replication bubble?

Answer 8

The open DNA that is being replicated

Question 9

What is meant by replication is typically bidirectional?

Answer 9

DNA replication occurs both towards and away from the replication fork, but always in the 5’-3’ direction

Question 10

What is meant by replication is semidiscontinuoys

Answer 10

because DNA strands must be synthesized in 5’-3’ direction and strands are antiparallel;

(1) leading strand is synthesized continuously (towards the fork)
(2) lagging strand is synthesized discontinuously (away from the fork) in a series of Okazaki fragments

Question 11

What groups are covalently linked in the 5’-3’ directionality

Answer 11

alpha-5’-phosphate of a new dNTP to the 3’-OH position of the nucleotide residue at the 3’ end

Question 12

What are RNA primers and what do they do?

Answer 12

They are short 10-13 base pair RNA complementary to the template. They are synthesized by primases and are NEEDED to begin synthses of DNA

Question 13

Which enzymes work together to replace RNA primers with DNA to seal remaining nick

Answer 13

nucleases, polymerases and ligases

Question 14

What are nucleases?

Answer 14

Enzymes that hydrolyze the internucleotides linkages of nucleic acid

Question 15

What are polymerases?

Answer 15

Enzymes that catalyzes template-dependent synthesis of DNA from its dNTP precursor

Question 16

What are ligases?

Answer 16

Enzymes that create a phosphodiester bond between the 3’ end of one DNA segment and the 5’ end of another

Question 17

Which polymerase seal the remaining nick once the RNA primers have been removed?

Answer 17

DNA polymerase I (DNA Pol I)

Question 18

What are exonucleases?

Answer 18

Enzymes that hydrolyze only those phosphodiester bond that are in a terminal positions of a nucleic acid

Question 19

Describe how DNA Pol I seals the nick

Answer 19

DNA Pol I degrades the RNA primer in he 5’-3’ direction, releasing dNMPs, and simultaneously extends the 3’ terminus with dNTPs in the same direction – ligase then links the fragments

Question 20

What are the 3 separate domains of DNA Pol I (not counting 3’-5’ exonuclese domain)

Answer 20

(1) Fingers; where dNTPs enter
(2) Thumb; holds the template DNA strand in place so that it can incorporate many nucleotides without disruption
(3) Palm; Active site of the enzyme where dNTPs attach

Question 21

What does DNA Pol I resemble?

Answer 21

a right hand

Question 22

What are the 2 conformations of DNA Pol I?

Answer 22

Open and closed

Question 23

Describe when DNA Pol I is open and when it is closed

Answer 23

(1) Begins in open form, the free 3’-OH is sitting in the active site and the fingers domain has contacted a dNTP
(2) It “closes” to bring the dNTP in proximity to the growing strand

Question 24

What does the accuracy of polymerase rely on?

Answer 24

Shape recognition involving H-bonding, van der Waals forces, and ionic bonding interactions. Incorrect base pairs for not fit well and prevent complete closure of DNA Pol I

Question 25

What are the 3 moieties component what have an important role in the stability and efficiency of the DNA synthesis reaction?

Answer 25

(1) Asp residues

(2) Mg ++ x 2

Question 26

What is the role of the Asp residues(in the active site of DNA Pol I) in the DNA synthesis reaction

Answer 26

They are acidic and therefore carry a negative charge at physiological pH
They don’t play a direct role in catalysis
They coordinate the 2 divalent Mg++

Question 27

What is the role of the 1st Mg++ (in DNA Pol I) in the DNA synthesis reaction?

Answer 27

Deprotonate the 3’OH group of the growing strand = 3’O nucleophile
3’O nucleophile is able to “attack” the alpha-Phosphate on the incoming dNTP
–this will form a phosphodiester bond and release pyrophosphate–

Question 28

What is the role of the 2nd Mg ++ ( inDNA Pol I) in the DNA synthesis reaction?

Answer 28

Responsible for bind the negatively charged phosphate group on the incoming dNTP, and facilitating the rapid release of the PPi leaving group after addition of the nucleotide (so the next one can enter the active site)

Question 29

Describe the steps of DNA polymerase reaction

Answer 29

(1) requirements; a primer strand, a template strand, dNTPs
(2) Active site has an postinsertion site and an insertion site because once the dNMP is incorporate PoL I moves forward to the new 3’terminus to incorporate another dNTP
(3) Insertion site contains template strand that will pair with incoming dNTP. Postinsertion site contains the 3’OH group that will attack the phosphodiester bond that connect the alpha and beta phosphates of the income dNTP. Addition of dNMP to the primer strand an release of pyrophosphate
(4) New terminal base is translocate from the insertion site to the postinsertion site, by sliding or dissociation of the enzyme from the DNA followed by rebinding with the terminal base pair in the postinsertion site

Question 30

Why is DNA Pol I able to slide along the template strand?

Answer 30

Several positively charged Lysine residues located within the palm domain interact with the negative phosphodiester backbone via electrostatic interactions

Question 31

What is processivity?

Answer 31

Ability to catalyze consecutive reactions without releasing its substrate - processivity number is the average number of nucleotides incorporated before the enzyme dissociates from the DNA

Question 32

How does the DNA Pol recognizes base mispairing?

Answer 32

Once a complementary nucleotide is added to the DNA chain, DNA Pol uses the new base as the site of subsequent addition – It can only make an addition if everything is lined up, if it isn’t DNA Pol cannot proceed because the key chemical groups won’t be in the right places for catalysis to occur

Question 33

How does DNA Pol correct mispairing?

Answer 33

Once mispairing is sensed, DNA Pol will fray the double stranded DNA by 4 nucleotides (3 correct +1 incorrect). This place the incorrect dNMP in the active site of the 3’-5’ exonuclease domain, which will cleave the incorrect dNMP

Question 34

What are the steps of proofreading of DNA Pol

Answer 34

(1) Mispairing happens
(2) Polymerase reposition the mispaired 3’OH terminus into the 3’-5’ exonuclease site by fraying the strand by 4 base pairs
(3) 3’4’ exonuclease hydrolyzes the mispaired base
(4) 3’OH terminute repositions back to the polymerase insertion site
(5) Polymerase incorporate correct nucleotide into the primer strand

Question 35

Describe the active site of 3’-5’ exonuclease

Answer 35

(1) One Mg++ ion deprotonates an H2O molecule to form an OH- nucleophile, which can attack the phosphate and mediate hydrolysis of the dNMP that is incorrect leaving a free 3’OH
(2) 2nd Mg++ ion promotes departure of the incorrectly paired dNMP from he growing strand

Question 36

Proofreading is not simply the reverse of the polymerization reaction, name one significant difference

Answer 36

DNA Pol starts with a dNTP and adds a dNMP while exonuclease cleaves and releases a dNMP

Question 37

What is a “nick”?

Answer 37

the gap between lagging strand fragments

Question 38

Describe the process of Nick Translation by Pol I

Answer 38

(1) At a Nick, Pol I degrades the RNA primer in the 5’-3’ direction
(2) This releases dNMPs and simultaneously extends the 3’ terminus with dNTPs in the same direction
(3) Net result is movement of the nick in the 5’-3’ direction along the DNA until all RNA is removed
(4) DNA ligase can then seal the fragments once Pol I dissociates

Question 39

What does the unique 5’-3’ exonuclease activity found in Pol I reflects?

Answer 39

The enzyme’s role in DNA repair, as it can perform a host of clean-up functions during replication, recombination and repair