DNA Structure & Replication

Question 1

what characteristic properties must the hereditary material have?

Answer 1

high capacity for information storage, chemically stable
replicate accurately
be capable of variation

Question 2

what seven sources prove that DNA is the hereditary material for life?

Answer 2

chromosome analysis
metabolic stability of DNA
constancy of DNA within a cell
correlation between mutagens and their effects on DNA
Griffith’s Experiment (DNA can transform bacteria)
Avery’s Experiment (transforming factor was DNA)
Hershey-Chase Experiment (DNA not proteins)

Question 3

what are nucleotides, and what are they composed of?

Answer 3

nucleotides are the base units of DNA or RNA, and are macromolecules that exist as polymers called polynucleotides

each nucleotide contains: a five carbon sugar (pentose), a nitrogenous base, a phosphate group

the pentose sugar can be either a deoxyribose or ribose sugar, making deoxyribonucleic or ribonucleic acids respectively

Question 4

how is the pentose sugar of a nucleotide bonded, and compare the differences between a ribose and deoxyribose pentose sugar

Answer 4

five-carbon sugars in ring form
- 5’ carbon to phosphate group (ester bond)
- anomeric 1’ carbon to nitrogenous base (glycosidic bond)

at 2’ of deoxyribose, there is an hydroxyl (OH) group
at 2’ of ribose, there is a H atom
partial negative charge of hydroxyl group in ribose repels negative charge of phosphate, so RNA cannot coil as tightly as DNA does (helix)
RNA more susceptible to chemical and enzyme degredation

Question 5

what are nitrogenous bases (two types: purine and pyrimidine, four bases), and the difference between T and U?

Answer 5

it has a nitrogen-containing ring structure

purines: 6-membered ring fused to 5-membered ring (adenine and guanine)
pyrimidine: 6-membered ring (cytosine and thymine / uracil)

the only difference between T and U is the presence of a methyl substituent at the fifth carbon

Question 6

what is a nucleoside, and how is it formed? what two types are there?

Answer 6

pentose + nitrogenous base = nucleoside

condensation reaction, pentose’s 1’ carbon bonded in glycosidic bond to nitrogenous base

two types: ribonucleosides and deoxyribonucleosides

Question 7

how are dinucleotides and polynucleotides formed?

Answer 7

condensation reaction: phosphodiester bond between the 5’-phosphate group of one nucleotide and the 3’-hydroxyl group
polynucleotide is made up of several million nucleotides, linear and unbranched sugar-phosphate backbone (strong covalent bonds conferring strength and stability)

Question 8

how is polarity / directionality conferred in a polynucleotide, and what are the two free ends of a polynucleotide chain like?

Answer 8

deoxyribonucleoside triphosphates are added to a growing chain in a manner that has polarity or directionality, since each end of the DNA or RNA strand has two chemically different free ends

5’ end: free 5’ carbon carrying a phosphate group
3’ end: free 3’ carbon carrying a hydroxyl (OH) group

DNA or RNA base sequence is read in a 5’ to 3’ direction

Question 9

what did X-ray diffraction conclude about DNA structure?

Answer 9

Maurice Wilkins and Rosalind Franklin: DNA structure using X-ray crystallography

diameter 2nm, 2-stranded, coiled in a double helix, one complete twist every 3.4nm, 10 bases to each complete turn

Question 10

what is Chargaff’s rule?

Answer 10

specific complementary base-pairing (amount of A = T and G = C)

Question 11

what are the main features of the DNA double-helix?

Answer 11

DNA consists of two polynucleotide strands / chains which are right-handed helixes coiled around each other to form double-helix
diameter of the helix is uniformly 2nm, space for 1 purine and 1 pyrimidine in the center
strands are anti-parallel
each has a sugar-phosphate backbone with:
- hydrophilic phosphate groups projecting out
- relatively hydrophobic nitrogenous bases orientated inwards towards central axis at almost right angles
bases of opposite strands bonded by relatively weak hydrogen bonds (two between AT, three between GC)

Question 12

how does complementary base pairing between DNA strands come about?

Answer 12

• A-T and C-G pairs can fit the physical dimensions of the double-helix
• in accord with Chargaff’s rule
• base sequence in one strand determines base sequence in the complementary strand
• relatively weak hydrogen bonds make it easy to separate the two strands of DNA

Question 13

what 5 features stabilise DNA double-helix (IMPT)?

Answer 13

• extensive hydrogen bonds between base pairs
• hydrophobic interactions between stacked base pairs
• exposure to outside influences of only sugar-phosphate backbone
• nitrogenous bases safely tucked inside double-helix
• (eukaryotes only) DNA tightly wound around histone proteins, forming repeating array of nucleosomes that fold into chromosomes, preventing thermal and physical damage

Question 14

what is semi-conservative DNA replication?

Answer 14

one parental DNA molecule makes two daughter DNA molecules (with one parental and one daughter each)

Question 15

how was the semi-conservative method of DNA replication proven by the Meselson-Stahl experiment?

Answer 15

only 15N heavy DNA e. coli
grew 1 generation on 14N
density-gradient centrifugation (in CsCl) performed

1st generation: all hybrid DNA (one heavy 15N, one light 14N)
2nd generation: 50% hybrid, 50% light

only attainable if replication is semi-conservative, so conservative and dispersive theories were disproven

Question 16

how many origins of replication are there in prokaryotes, and why?

Answer 16

single oriR since prokaryotic chromosome is a small circular DNA molecule
DNA replication proceeds bidirectionally from oriR to termination site around halfway around circular chromosome (synthesising two daughter DNA molecules)

Question 17

how many origins of replication are there in eukaryotic cells, and why is this so?

Answer 17

eukaryotic chromosome is much larger and has a linear DNA molecule
multiple oriRs
speed: multiple replication bubbles form and eventually fuse, speeding up the coping of very long DNA molecules, shortening it by 100 times

Question 18

what is the role of helicases in DNA replication? (memorise)

Answer 18

using ATP, break H bonds btw DNA strands, unwinding and separating parental strands

Question 19

what is the role of SSB proteins in DNA replication? (memorise)

Answer 19

SSBs stabilise single-stranded DNA binding proteins, preventing ssDNA from re-annealing

Question 20

why are RNA primers needed?

Answer 20

none of the DNA polymerases can initiate the synthesis of a DNA strand on its own

solution: primase (an RNA primer) hydrolyses ATP to produce a primer (of about 10 nucleotides long in eukaryotes) with a free 3’ OH end that DNA polymerase can extend
the primer is later replaced by a DNA polymerase with 5’ to 3’ exonuclease activity that swaps them for DNA nucleotides

Question 21

how is accurate complementary base-pairing ensured, and what is DNA polymerase’s role in this process?

Answer 21

complementary base-pairing between templates and nucleotides: the template parental strand is read by DNA polymerase, which assembles the deoxyribonucleoside triphosphate monomers for the newly-synthesised daughter strand
if an incorrect base pair is recognised, DNA polymerase reverses its direction by one base pair of DNA, and the 3’ to 5’ exonuclease activity of the enzyme allows the incorrect base pair to be excised (proofreading)

Question 22

how are the incoming DNA nucleotides bonded to the growing daughter DNA strand?

Answer 22

DNA polymerases catalyse phosphodiester bond formation between the free 3’ hydroxyl (OH) group of the last nucleotide in the growing strand and the free 5’ phosphate group of the incoming dNTP
the incoming dNTP loses a pyrophosphate group, and energy released from that bond breakage is coupled to phosphoester bond formation

Question 23

what is leading and lagging strand synthesis?

Answer 23

leading: complementary daughter DNA that is continuously synthesised

lagging: discontinuously synthesised in Okazaki fragments

Question 24

what is the end replication problem in linear eukaryotic chromosomes? (memorise)

Answer 24

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

Question 25

summarise DNA packing

Answer 25

• DNA double helix coils around histone proteins, forming 2nm nucleosomes
• nucleosomes joined by linker DNA, forms 10nm “beads on string”
• 10nm chromatin coils into 30nm solenoid with bonds between linker DNA and histone tails
• 30nm chromatin attached at multiple points on protein scaffold to form looped domains of 300nm chromatin fibre

Question 26

summarise DNA synthesis (3 points, memorise)

Answer 26

1. DNA polymerase selects dNTPs complementary to template
2. catalyse formation of phosphodiester bonds to join incoming nucleotides to free 3’OH group
3. DNA polymerase proofreads

Question 27

what do the two enzymes do in lagging strand synthesis?

Answer 27

DNA polymerase I removes RNA primers, replaces with dNTPs

DNA ligase catalyses formation of phosphoester bond between 3’ of new Okazaki and 5’ of growing daughter strand (NOT phosphodiester)