Structure of DNA and DNA replication

Question 1

the natural form of DNA

Answer 1

dsDNA, double stranded DNA.

It is 2 polynucleotide strands wrapped around each other in a double helix

Question 2

DNA name

Answer 2

Deoxyribonucleic acid. It is a nucleotide polymer

Question 3

What is a nucleotide

Answer 3

A phosphate group PO4-

Pentose sugar

A nitrogenous base

The carbon atoms of the five-carbon deoxyribose are numbered 1ʹ, 2ʹ, 3ʹ, 4ʹ, and 5’

A nucleotide is simply a nucleoside with additional phosphate group or groups.

Question 4

What is 5ʹ-3ʹ phosphodiester bonds

Answer 4

Links 2 nucleoside triphosphate together between the 3’ carbon and the 5’ carbon of the next nucleotide.

Phosphodiester bonding between nucleotides forms the sugar-phosphate backbone.

Question 5

What is dNTP and describe the phosphate groups

Answer 5

Nucleoside triphosphate, a nucleotide which has a deoxyribose sugar, a nitrogenous base and 3 phosphate groups.

Phosphate groups are bound to the 5 prime carbon of the ribose sugar. The 5 prime carbon is the CH2, CH2 is bound to a oxygen of the PO4.

For each PO4, there is a negative charge and 1 oxygen is double bonded to phosphate. The final PO4 has -2 charge on both oxygen atoms with no double bonds.

The nitrogenous base is bound to the 1 prime carbon of the ribose sugar via a 1’ glycosidic bond.

Alpha, beta and gamma phosphate groups are the 1,2,3 phosphate groups respectively.

Question 6

What are purines?

Answer 6

Purines are double ringed. They are adenine and guanine.

Question 7

What are pyrimidines?

Answer 7

Pyrimidines are single ringed, They are uracil, cytosine and thymine. Uracil replaced thymine in RNA.

Question 8

What is complimentary hydrogen bonding?

Answer 8

G and C bonds complimentarily via 3 hydrogen bonds.

A and T bond complimentarily via 2 hydrogen bonds.

Question 9

What is a nucleoside

Answer 9

Pentose + nitrogenous base

Question 10

What are the 4 key features of DNA

Answer 10

1. Backbone of helix is composed of two chains with alternating sugar and phosphate units
2. Bases in opposite strands hydrogen bonds according to ATGC rule
3. 2 strands are antiparallel
4. There are approximately 10 nucleotides in each strand per complete turn of the helix
5. A complete turn is 3.4nm long
6. Nitrogenous bases lie perpendicular to central axis, forming the “rungs” of the twisted ladder
7. Alternating major and minor grooves

Question 11

List the 2 types of nucleic acids

Answer 11

DNA
- Hereditary molecule (carries genetic material)

RNA
- Certain viruses (HIV, Coronavirus, Influence virus) use RNA as carrier of genetic information

Question 12

List 6 difference of RNA from DNA

Answer 12

RNA contains ribose sugar whereas DNA contain deoxyribose sugar. Ribose has a OH on the 2’ carbon whereas deoxyribose has no OH on the 2’ carbon.

RNA uses uracil whereas DNA uses thymine.

RNA is commonly found in cytoplasm whereas DNA is commonly found in cell nuclei.

RNA is usually shorter than DNA

RNA is usually single stranded whereas DNA is usually double stranded.

RNA has 3 types, mRNA, tRNA and rRNA whereas DNA has 1 type.

Question 13

List 4 function of DNA

Answer 13

-  Storage of information
Most cell contain a complete set of genetic information, only a part of this genetic information is expressed at any given point.
-  Expression of information
-  Replication
-  Variation by mutation

Question 14

Describe the flow of genetic information

Answer 14

DNA -> Transcription -> mRNA + tRNA + rRNA (ribosomes) -> translation -> Protein

Question 15

List the 2 types of cellular division

Answer 15

Mitosis - formation of diploid daughter cells, cellular replication

Meiosis - formation of gametes, cellular reproduction

Question 16

Describe the steps in human DNA replication

Answer 16

Stage 1: Strand separation
DNA helicase breaks the hydrogen bonds between complimentary base pairs of the DNA. This creates a replication fork

1 of the strand is oriented in the 3’ to 5’ direction, towards the replication fork also known as leading strand. The other strand is oriented in the 5’ to 3’ direction, away from the replication fork also known as the lagging strand.

SSBP bind and stabilize the fork, prevent renaturation of DNA.
Gyrase relax the supercoiling and create nicks to allow coiling to occur.

Stage 2: Strand elongation

For the leading strand
A RNA primer, produced by primase comes along and bind to the leading strand. The primer acts as the starting point of DNA synthesis.

DNA polymerase 3 binds to the leading strand and goes along the leading strand to produce new complimentary molecules in the 5’ to 3’ direction.
This type of replication is continuous

For the lagging strand
Numerous RNA primers made by the primase binds at various points along the lagging strand.
Okazaki fragments are added to the lagging strand in the 5’ to 3’ direction.
This replication is discontinuous as the fragments have to be joined up later,

Stage 3: Termination
Once all bases are matched up, exonuclease removes the primers, DNA polymerase 1 fill the gap left by the primers with more complimentary molecules and proofread the new DNA to ensure there is no mistakes.
DNA ligase seals up the sequences of DNA into 2 continuous double strands.
The result is 2 DNA molecules consisting of 1 old and 1 new chains of nucleotides. Hence, DNA replication is semi-conservative as half of the new DNA template comes from the original DNA strand.
The DNA automatically rewinds into a double helix.

Question 17

List the 5 types and general functions of the 5 E.coli DNA polymerases

Answer 17

1: Removes RNA primers and fills gap with
DNA (i.e. processing of Okazaki
fragments)
2: Participates in DNA repair
3: Replicates DNA and does
proof-reading
4 and 5: – Specialized DNA polymerases,
participates in DNA repair and replicating of damaged DNA

Question 18

List the 3 raw materials for DNA synthesis

Answer 18

• > Free deoxyribonucleoside triphosphates (dNTPs)
• dATP, dTTP, dGTP, dCTP

-> Enzymes - e.g. DNA polymerase I and III, helicases,
ligase, gyrase

-> Template DNA - provides the base sequence that
specifies the complementary sequence of the new
DNA chain

Question 19

Helicase

Answer 19

• Helicase unwind the DNA double helix at ori by breaking the hydrogen bonds and thus denatures the DNA, use ATP as energy source.

Question 20

Topoisomerase

Answer 20

• Topoisomerase relax supercoiling of DNA in direction of replication fork, It also makes temporary single or double-stranded nicks in DNA backbone which allows uncoiling to occur, and subsequently repairs the nicks

Question 21

Single stranded binding proteins

Answer 21

• Single stranded binding proteins bind to denatured single-stranded DNA to stabilize the open structure of replication fork and prevent strand renaturation

Question 22

DNA polymerase 3

Answer 22

• > Requires a free 3’ OH end (i.e. from the short RNA primer synthesized by Primase)
• > Elongates the RNA primer by adding complementary bases in the 5’ to 3’ direction, using the parent DNA strand as a template
• > Proof-reading function i.e. a 3’ to 5’ exonuclease activity capable of detecting and excising mismatched bases on newly synthesized DNA strands
• > Fidelity of DNA replication - with only one error in every 100 million bases duplicated

Question 23

DNA polymerase 1

Answer 23

• > Removes the RNA primer (5’ to 3’ exonuclease activity) and fills the gaps with complementary DNA
• > It is a template-dependent enzyme - it only adds nucleotides that correctly base pair with an existing DNA strand acting as a template

Question 24

Ligase

Answer 24

• > Closes any nicks in the DNA backbone by catalyzing the formation of a phosphodiester bond between the adjacent 3’ OH and 5’ P groups
• > Joins Okazaki fragments together into a continuous strand of DNA

Question 25

Primase

Answer 25

-> Synthesize short RNA primer (5-15 bases long) on leading and lagging strands, using DNA as a template

-> Provides a free 3’ OH end for DNA polymerase III
(Primase does not require a free 3’ OH end to initiate RNA primer synthesis)

Question 26

Continuous synthesis

Answer 26

The other strand (leading strand) is synthesized by continuous addition of nucleotides to the growing end.

Question 27

Discontinuous synthesis

Answer 27

The synthesis of a new strand of a replicating DNA molecule as a series of short fragments that are subsequently joined together.

Question 28

Problems with DNA replication

Answer 28

-> Two strands of the double helix are anti-parallel to
each other (i.e. 5’ 3’ and 3’ 5’)
-> DNA polymerase III can only elongate DNA in the 5’
to 3’ direction
-> Thus, DNA polymerase III catalyzes two types of DNA synthesis reactions

• > The leading strand is made continuously in a 5’ to 3’ direction from the origin
• > The lagging strand is made discontinuously in a 5’ to 3’direction to form Okazaki fragments

Hence, a single RNA primer is sufficient for the
continuous replication of the leading strand

• But the discontinuous replication of the lagging
strand requires multiple primers, an RNA primer to
start the synthesis of each Okazaki fragment

• DNA polymerase I removes the RNA primer and
replaces the missing nucleotides

• The Okazaki fragments are later joined together by
DNA ligase which forms the phosphodiester bond
between the fragments

Question 29

Short summary of DNA replication

Answer 29

Helicase unwinds and separates the DNA strands so that replication can start

1. Single-stranded binding proteins keep the DNA apart
2. DNA gyrase relaxes supercoiling of DNA
3. Primase synthesizes RNA primers required by DNA Polymerase III
4. DNA Polymerase III synthesizes the new DNA strands
5. DNA Polymerase I removes the RNA primer and replaces it with DNA
6. DNA Ligase joins the Okazaki fragments together

Question 30

DNA polymerase

Answer 30

DNA polymerases catalyze the formation of polynucleotide chains through the addition of successive nucleotides derived from deoxynucleoside triphosphates.

Question 31

DNA and RNA reading

Answer 31

Read from 5 prime to 3 prime, nucleotides are added at the 3 prime end.