Topic 7.1: DNA Structure and Replication

Question 1

Harshey and Chase aim

Answer 1

Determine if DNA or proteins were the genetic material of a cell

Question 2

Harshey and Chase experiment

Answer 2

1) It was previously known that viruses insert their genetic material into cells and so radioactively labeled viruses were prepared
2) Viruses grown in 35S had radioactive proteins but did not transfer this radioactivity to bacterium (remained in supernatant)
3) Viruses grown in 32P had radioactive DNA and did transfer this radioactivity to infected bacterium (found in pellet)

Question 3

X-ray Diffraction by Franklin and Wilkins

Answer 3

1) X-rays will diffract when targeted at crystallised DNA molecules
2) The scattering pattern created can be used to determine structure

Question 4

Deduced properties from X-ray diffraction

Answer 4

1) Composition: DNA is a double-stranded molecule
2) Orientation: The bases face inwards and the phosphates face out
3) Shape: DNA forms a double helix (10 bases per twist | 34 armstrong)

Question 5

DNA Structure (4)

Answer 5

1) Phosphates form an outer backbone and nitrogenous bases are packaged within the interior
2) DNA is composed of an equal number of purines and pyrimidines
3) Two strands must run in antiparallel directions
4) A–T bond was the same length as a G–C bond

Question 6

Deduction of DNA replication from DNA structure (2)

Answer 6

1) Replication occurs via complementary base pairing (A - T | G - C)
2) Replication is bidirectional (proceeds in opposite directions on the two strands) due to the antiparallel nature of the strands

Question 7

Nucleosomes

Answer 7

1) DNA
2) 8 Histones
3) H1

Question 8

Purpose of Nucleosomes

Answer 8

1) Makes DNA compact (better storage)
2) Prevents DNA damage (less exposed)
3) Assists in cell division (more mobility)
4) Involved in transcriptional regulation
5) Help to supercoil the DNA.

Question 9

Eukaryotic Organization of DNA (5)

Answer 9

1) Nucleosomes are linked to form a string of chromatosomes
2) Chromatosomes coil to form a solenoid structure (~6 chromatosomes per turn)
3) Solenoid is condensed to form a 30 nm fibre
4) 30 nm fibres form loops, which are compressed and folded to form chromatin
5) Chromatin supercoils during cell division to form chromosomes

Question 10

Non-Coding DNA

Answer 10

1) Satellite DNA (tandem repeats)
2) Telomeres (chromosome ends)
3) Introns (non-coding sequences)
4) Non-coding RNA genes
5) Gene regulatory sequences

Question 11

Tandem repeats

Answer 11

Long stretches of DNA made up of repeating element

Question 12

DNA Profiling

Answer 12

Technique by which individuals can be identified and compared via their respective DNA profiles

Question 13

DNA Replication Enzymes

Answer 13

1) Helicase
2) DNA Gyrase
3) Single Stranded Binding Proteins
4) DNA Primase
5) DNA Polymerase III
6) DNA Polymerase I
7) DNA Ligase

Question 14

Helicase (2)

Answer 14

1) Separates the DNA strands to form a replication fork

2) Breaks the hydrogen bonds between complementary base pairs

Question 15

DNA Gyrase (2)

Answer 15

1) Reduces the torsional strain created by helicase

2) Prevents the DNA from supercoiling as it is being unwound

Question 16

Single Stranded Binding (SSB) Proteins

Answer 16

Prevent DNA strands from reannealing

Question 17

DNA Primase (2)

Answer 17

1) Generates a short RNA primer on each strand
2) Primers provide an initiation point for DNA polymerase III (DNA pol III can only add nucleotides to 3’-end of a primer)

Question 18

DNA Polymerase III

Answer 18

1) Free nucleotides (dNTPs) line up opposite complementary bases
2) DNA polymerase III covalently joins free nucleotides together

Question 19

DNA Polymerase I

Answer 19

Removes RNA primers and replaces them with DNA

Question 20

DNA Ligase

Answer 20

Covalently joins the Okazaki fragments together

Question 21

Okazaki Fragments

Answer 21

Discontinuous segments of DNA

Question 22

Leading and lagging strand

Answer 22

1) On the leading strand, DNA pol is moving towards the replication fork and so can copy CONTINUOUSLY
2) On the lagging strand, DNA pol is moving away from the replication fork, meaning copying is DISCONTINUOUS.

Question 23

DNA Sequencing

Answer 23

Process by which the base order of a nucleotide sequence is elucidated

Question 24

Dideoxynucleotides

Answer 24

1) Lack the 3’-hydroxyl group necessary for forming a phosphodiester bond
2) Prevent further elongation of a nucleotide chain

Question 25

DNA Sequencing Process

Answer 25

1) Four PCR mixtures are prepared – each with stocks of normal bases and one dideoxynucleotide (ddA, ddT, ddG, ddC)
2) Whenever the dideoxynucleotide is randomly incorporated, the DNA sequence is terminated at that base position
3) Because a complete PCR cycle generates millions of sequences, every base position is likely to have been terminated
4) These sequences are separated by gel electrophoresis to determine base sequence (according to ascending sequence length)
5) Automated machines can determine the sequence quickly if fluorescent labeling of the dideoxynucleotides has occurred