dna structure & replication

Question 1

what is a nucleotide composed of?

Answer 1

a pentose (five-carbon) sugar
a nitrogenous base
a phosphate group

Question 2

what is a nucleoside composed of?

Answer 2

a pentose sugar
a nitrogenous base

Question 3

what are the 4 nitrogenous bases?

Answer 3

adenine, thymine, guanine, cytosine

Question 4

what is the difference between a deoxyribose sugar and a ribose sugar?

Answer 4

at the 2’ carbon, there is a hydrogen atom (H) in deoxyribose. whereas for ribose, at the 2’ carbon, there is a hydroxyl group (-OH).

the partial negative charge of the hydroxyl group in ribose repels the negative charge of the phosphate, preventing the RNA chain from coiling in as tight a helix as it does in DNA, so RNA is less stable than DNA and more susceptible to chemical and enzyme degradation

Question 5

what are the purine bases?

Answer 5

adenine & guanine

Question 6

what are the pyrimidine bases?

Answer 6

thymine, cytosine, uracil

Question 7

how is a nucleoside formed?

Answer 7

a pentose and a nitrogenous base is linked together through a condensation reaction where a water molecule is lost. the 1’ carbon is linked in a glycosidic bond to the nitrogenous base

Question 8

how is a nucleotide formed?

Answer 8

a nucleotide is formed by a condensation reaction between the nucleoside (pentose + nitrogenous base) and phosphate group, forming a phosphoester bond between the 5’ carbon of pentose and the phosphate group.

Question 9

how are nucleotide chains, or polynucleotides, formed?

Answer 9

1. 2 nucleotides join to form a dinucleotide by condensation between the 5’ phosphate group of one nucleotide and the 3’ hydroxyl group of the other to form a phosphodiester bond.
2. the condensation reaction is repeated several million times to form a polynucleotide
3. phosphodiester bonds between 5’ phosphate and 3’ hydroxyl groups of nucleotides form a linear, unbranched sugar-phosphate backbone
4. phosphodiester bonds are strong covalent bonds that confer strength and stability on the polynucleotide chain

Question 9

what does chargaff’s rule state about complementary base pairing?

Answer 9

1. the is always equal proportion of adenine & thymine and equal proportion of guanine & cytosine
2. there is always an equal proportion of purines and pyrimidines (i.e. A+G = 50%, C+T = 50%)

Question 10

why do nitrogenous bases form complementary base pairs?

Answer 10

because of steric restrictions & hydrogen bond factors.

steric restrictions: pyrimidines have a single ring while purines are about twice as wide as pyrimidines, so purines should be paired with pyrimidines for the double helix to have a uniform 2nm diameter
hydrogen bond factors: A is capable of forming 2 hydrogen bonds with T, while G is capabale of forming 3 hydrogen bonds with C

Question 11

describe the semi-conservative model of DNA replication

Answer 11

each of the2 daughter DNA molecules consists of one parental DNA strand and one newly-synthesised daughter DNA strand.

Question 12

DNA separation

what is the function of helicases?

Answer 12

helicases bind to one strand of the DNA molecule, and uses ATP as an energy source to break the hydrogen bonds holding the two strands of DNA together.
this unwinds the DNA double helix and separates the parental DNA strands at the region of the replication fork.
each of the 2 parental DNA strands serve as the template for the synthesis of a new DNA strand.

Question 13

DNA separation

what are the functions of single-strand DNA-binding proteins (SSB proteins)?

Answer 13

1. SSB proteins temporarily stablises unwound single-stranded portion of the DNA double-helix by binding onto it. this prevents the ssDNA from reannealing to reform the duplex, keeping the 2 parental strands in the approporiate single-stranded condition to act as template
2. protects ssDNA, which is very unstable, from being degraded.

Question 14

DNA separation

what are the functions of topoisomerases?

Answer 14

topoisomerases cleave a strand of the helix to create a transient single-stranded nick. this relieves strain on the DNA molecule, which is tense due to supercoiling ahead of the replication fork during unwinding, by allowing free rotation around the intact strand and then reseals the broken strand.

Question 15

what are the 2 limitations of DNA polymerases?

Answer 15

1. none of the DNA polymerases can initiate the synthesis of a DNA strand on its own.
2. DNA polymerases only add dNTPs to the free 3’ OH end of a growing DNA strand, and never to the 5’ end. a growing DNA strand can thus only elongate in the 5’to 3’ direction

2nd limitation makes continuous synthesis of both DNA strands impossible since the 2 strands of a DNA double helix are antiparallel

Question 16

how is the synthesis of a DNA strand, using an RNA primer, initiated?

Answer 16

1. a portion of the parental DNA strand serves as template for making the RNA primer with the complementary base sequnce
2. an enzyme called primase joins the ribonucleotides to make the primer, which is about 10 nucleotides long in eukaryotes. hydrolysis of ATP is involved in this.
3. the RNA primer provides a free 3’ OH end that DNA polymerase can extend, thereby priming the synthesis of the daughter DNA strand.
4. a DNA polymerase with 5’ to 3’ exonuclease activity later replaces the RNA nucleotides of the primers with DNA versions

Question 17

how are daughter DNA strands synthesised?

Answer 17

1. complentary base-pairing between templates & nucleotides
   - parental DNA strands, separated at the replication fork and each primed with an RNA primer, serve as the templates for semi-conservative DNA replication
   - DNA polymerase reads the template and assembles the dNTPs for the newly-synthesised daughter DNA strand based on complementary base-pairing
   - when an incorrect base pair is recognised, DNA polymerase reverses its direction by one base pair of DNA. proofreading is carried out by exonuclease, an enzyme, in the 3’->5’ direction (opposite to daughter strand synthesis direction) allows incorrect base pairs to be excised.
2. phosphodiester bond formation between growing daughter DNA strand and incoming nucleotide
   - DNA polymerases catalyse the polymerisation of the daughter DNA strand
   - all DNA polymerases catalyse phosphodiester bond formation between a growing daughter DNA strand and an incoming nucleotide
   - because of active site specificity of the DNA polymerases, syntheses of both daughter DNA strands can only occur in one direction (5’->3’)
   - addition of dNTP requires a phosphoester bond to be formed between the free 3’ OH group of the last nucleotide in the growing strand and the free 5’ phosphate group of the incoming dNTP
   - dNTP loses a pyrophosphate group in the process.

Question 18

what is the leading strand?

Answer 18

the leading strand is the complementary daughter DNA strand that is continuously synthesised as a single polymer along the template strand.
it is polymerised in the 5’->3’ manner towards the replication fork

Question 19

what is the lagging strand?

Answer 19

the lagging strand is the complementary DNA strand that is discontinuously synthesised as a series of short fragments known as okazaki fragments.
each okazaki fragment is polymerised in the mandatory 5’->3’ direction AGAINST the overall direction of the replication fork

for the lagging strand, the template strand of DNA used is extending with the replication fork in the 3’->5’ direction. so since daughter DNA strands are synthesised in the 5’->3’ direction, the daughter DNA strands cannot be synthesised continuously since the replication bubble keeps expanding

Question 20

how are okazaki fragments joined to form a continuous DNA strand

Answer 20

each okazaki fragment requires an RNA primer for strand initiation.
the okazaki fragments are then ligated in 2 steps to produce a continuous DNA strand:
1. DNA polymerase removes the RNA primer and replaces it with dNTPs
2. DNA ligase catalyses the formation of a phosphoester bond between the 3’ end of each new okazaki fragment and the 5’ end of the growing daughter DNA strand.

Question 21

what is the end replication problem?

Answer 21

the end replication problem occurs in linear chromosomes as DNA polymerase is incapable of completely replicating all the way to the ends of linear chromosomes, leading to shortening of telomeres.

Question 22

why does the end replication problem occur?

Answer 22

the very end of the lagging strand is not replicated.
1. a small section at the extreme 3’ end of the parental strand does not undergo DNA replication during cell division
2. bc when the final RNA primer at the end of the lagging strand is removed, there is no upstream strand onto which DNA polymerase can build to fill the resulting gap
3. the daughter DNA strand will be shortened with each round of replication