DNA structure and replication

Question 1

What evidence is there that DNA is the genetic material?

Answer 1

Griffith, 1928:
Studied the transformation of pneumococci in vivo = information can be transferred between different strains of bacteria

Hershey & Chase, 1952:
Studied bacteriophage infection
= DNA can be transferred to infect other bacteria = DNA is the genetic material not protein

Question 2

Phage =

Answer 2

Virus that infects bacteria and multiply inside the cells causing lysis which releases progeny phage to infect other bacteria

Question 3

Purine =

Answer 3

• Larger
• Composed of 2 fused rings incorporating 2 nitrogen atoms in each ring
• Adenine and Guanine

Question 4

Pyrimidines =

Answer 4

• Smaller
• Single ring with 2 nitrogen atoms
• Thymine, Cytosine, Uracil

Question 5

Nucleotide =

Answer 5

Nucleoside + Phosphoric acid

Question 6

Nucleoside =

Answer 6

Nitrogen base + Pentose sugar

Question 7

Central Dogma

Answer 7

DNA > transcription > RNA > translation > Protein

Question 8

Enzymes that manipulate DNA

Answer 8

• Nucleases
• Ligases
• Polymerases
• Kinases + phosphatases
• Methylases + demethylases
• recombinases + topisomerases

Question 9

When DNA is melted …

Answer 9

Two strands are separated by thermal vibration - disrupts hydrogen bonds that holds strands together

Question 10

Anneal/hybridise =

Answer 10

DNA strands joined back together during slow cooling

Question 11

Endonuclease -

Answer 11

Cuts double stranded DNA at internal sites

= sticky ends (staggered cut) or blunt ends (straight cut)

Question 12

Exonucleases -

Answer 12

Cuts DNA strands at the end

Question 13

Ligate =

Answer 13

Join together two DNA fragments that have been cut by the same enzyme = recombinant DNA

Question 14

Restriction endonucleases =

Answer 14

• enzymes used to generate DNA fragments for subsequent re-assortment, ligation and cloning
• recognise + chop DNA
• protects microorganisms from foreign DNA by recognising inverted repeats (palindromes) because they bind to DNA as diners

Question 15

Example of an restriction endonuclease…

Answer 15

Thermostable DNA polymerase is used in the polymerase chain reaction

Question 16

Polymerase chain reaction =

Answer 16

An artificial method for multiplying fragments of DNA

Makes many copies of a specific sequence of DNA in vitro

Question 17

To carry out the polymerase chain reaction you need…

Answer 17

• DNA primers
• Taq polymerase
• free nucleotides
• Buffer

Question 18

Steps of the polymerase chain reaction…

Answer 18

1) denaturation - 96 degrees Celsius = splits DNA into single strands
2) annealing - 55 degrees Celsius = primers bind to single strand, this creates a small section of double stranded DNA
3) extension - 72 degrees Celsius = taq polymerase binds to double stranded DNA and adds free nucleotides

Question 19

Gene cloning =

Answer 19

Isolating DNA from one organism and propagating it in another organism to produce a genetically identical copy

Question 20

Cloning vector =

Answer 20

A small piece of DNA, taken from a virus/plasmid that has a foreign DNA fragment inserted in it - can insert DNA into a host cell

Question 21

Plasmid =

Answer 21

Small DNA molecule within a cell that is physically separated from a chromosomal DNA and can replicate independently

Question 22

Ligation =

Answer 22

The joining of two DNA strands by a phosphate Ester linkage

• used to form circular recombinant plasmids

Question 23

Transformation =

Answer 23

Alteration of the genetics of a cell by the direct uptake and expression of DNA from its surroundings

• how recombinant plasmids are introduced into host cells

Question 24

Sanger Method of gene cloning…

Answer 24

• gene that is being sequenced is cut out of the DNA strand using restriction enzymes
• gene is inserted into a vector (plasmid)
• plasmid is taken up by bacterium
• when bacteria divides by binary fission the plasmid with the gene is copied

Question 25

Meselson & Stahl, 1958…

Answer 25

…Proved that DNA replication proceeds via a semi-conservative mechanism using density gradient centrifugation - used light Nitrogen-14 and heavy Nitrogen-15 which form separate bands at positions corresponding to their buoyant densities. - found that the strands separate and each act as a template for synthesis

Question 26

Requirements of DNA polymerase 1…

Answer 26

- dNTPs plus Mg2+ - template - primer with 3’-OH

Question 27

All DNA polymerases…

Answer 27

- require dNTPS plus Mg2+, DNA templates and primers - synthesise in the 5’ > 3’ direction - have 3’ > 5’ exonuclease activity - processive

Question 28

Differences between DNA polymerases…

Answer 28

-polymerase 1 and 2 are slow but polymerase 3 is faster

Question 29

DNA polymerase 1 -

Answer 29

A single polypeptide chain with 3 structural domains Makes large rRNAs

Question 30

DNA polymerase 3-

Answer 30

Multimeric enzyme in which specific properties are contributed by different subunits Makes tRNAs

Question 31

Replication of E.coli chromosome…

Answer 31

- E.coli chromosome is circular - bidirectional replication starts at origin site - two replication forks assemble at origin site and move away from each other

Question 32

Replication fork=

Answer 32

A region of DNA undergoing simultaneous unwinding, strand separation and replication

Question 33

Potential problem in DNA replication - polarity:

Answer 33

All 3 DNA polymerases work only in the 5’ > 3’ direction but DNA is antiparallel = Okazaki model - one strand is synthesised continuously and one is synthesised discontinuously

Question 34

Okazaki fragments =

Answer 34

Fragments of DNA that are produced during DNA replication when the lagging strand is copied piece by piece (discontinuously)

Question 35

Potential problem in DNA replication - primers:

Answer 35

DNA polymerases need primers to be able to attach DNA nucleotides to the template strand Primer = a short strand of DNA that serves as a starting point for DNA synthesis - primers are synthesised by primase

Question 36

Potential problem in DNA replication - need to seal nicks:

Answer 36

DNA polymerase cannot seal nicks in the phosphodiester backbone that is left following primer removal - DNA ligase seals together nicks and patches Okazaki fragments together

Question 37

Potential problems in DNA replication - topology:

Answer 37

- Helicase breaks hydrogen bonds = unwinds DNA - Single stranded DNA binding proteins = stops single strands of DNA from forming hydrogen bonds - DNA topoisomerases = control superhelical density

Question 38

DNA topoisomerases =

Answer 38

Control superhelical density of DNA - binds to DNA and cuts phosphate backbone to allow it to untangle E.g. DNA gyrase = removes positive supercoils

Question 39

Primase =

Answer 39

A multi-protein complex that recognises the sites where the origin is and as the for moves away also recognises the sites where primers should be laid down for the Okazaki fragments of the lagging strand.

Question 40

Leading strand =

Answer 40

Synthesised continuously along the entire chromosome

Question 41

Lagging strand =

Answer 41

Discontinuously synthesised - forms Okazaki fragments

Question 42

Promoter =

Answer 42

A piece of double stranded DNA Specific binding site for RNA polymerase Directional Determines which strand is replicated

Question 43

DNA polymerase 2-

Answer 43

Transcribes protein-encoding genes in eukaryotes Makes mRNA

Question 44

Number of amino acids represented in the genetic code =

Answer 44

20