Lecture 13

Question 1

the double helix

Answer 1

two nucleotide polymers
bound by complementary base pairing
phosphodiester bonds have 5’-3’ polarity
antiparallel strands
helical structure with specific features

Question 2

Structural challenges of replication

Answer 2

how the new and old stands generate two new double helices

how are the nucleotides exposed and read

how to ensure fidelity

how to organise the enzymatic activities for multiple long DNA molecules

Question 3

Conservative replication

Answer 3

histone scaffold

breaks hydrogen bonds

new nucleotide pairing without unwinding

need an isomerase activity to turn bases respect deoxyribose

Question 4

Semi-conservative replication

Answer 4

breaks hydrogen bonds

new double helices are assembled as replication progresses

needs extensive unwinding: potential need for helicases

Question 5

dispersive replication

Answer 5

no unwinding required
swapping templates strands
but requires multiple single-stranded breaks
assumes polymerisation is in both directions

Question 6

Taylor, Woods and Hughes

Answer 6

growing root tip of vicia faba

they supplied thymidine labelled with tritium and after labelling the chromosomes incubate in cold medium with colchicine (blocks cytokinesis)

they looked at cells in metaphase and observed distribution of thymidine in chromosomes

Question 7

Meselson and Stahl

Answer 7

reasoned that DNA with 15N in its nitrogens bases would be denser than with 14N so should lead to different sedimentations behaviour under CsCl gradient centrifugation

conc of Cs ions would be different along the test tube so DNA would sit at different heights based on density

Question 8

Features of semi-conservative replication

Answer 8

fast
- human genome replicated in around 8 hours
accurate
-error rates of 10-5 in human polymerases but DNA repair mechanisms lower the overall rates to estimate 10-8
not perfect
- some errors , 7-0 new mutations per generation
Highly regulated
- dozens of enzymes participate in the process

Question 9

Replication initiated at origins

Answer 9

replication proceeds in both ends of the bubble (forks)
however polymerisation is in one direction only

the replication bubble requires unwinding the strands of DNA

a eukaryotic chromomse may have 100s-1000s of origins of replications, this increases speed

Question 10

replication forks

Answer 10

a very active area where DNA replication takes place

Question 11

How do replication forks advance

Answer 11

1. requires finding the 3-5 DNA polymerase
   or
2. requires finding the small fragments and explaining how they are joined

Question 12

Okazakis experiments

Answer 12

they used the replication of a phage in E.coli

they used sedimentation to separate DNA in this case of different lengths

they labelled the newly synthesised DNA with thymidine and then measured radioactivity in the distribution of sizes

then they used mutant phages with temperature-sensitive ligase (to join small fragments)

they did many other experiments using 5-3 exonuclease to prove that newly added nucleotides were only incorporated at the 3’ ends

Question 13

leading strands

Answer 13

can be synthesised in a continuous process

Question 14

lagging strands

Answer 14

the lagging strand requires additional steps

Question 15

Essential enzymatic activities

Answer 15

DNA polymerase
can only synthesise in 5-3 orientation
needs a primer that provides 3 end

DNA primases
synthesise 5-10nt RNA primers from scratch using DNA as a template

Helices
unwind the double helix at the replication forks

Topoisomerases
relieve the strain of twisting of the double helix by breaking, swivelling and rejoining DNA strands ahead of the fork

Question 16

Synthesis of the leading strands

Answer 16

as the fork advances the leading strand keeps elongating

Question 17

Synthesis of the lagging strand

Answer 17

like with the leading strand first the primase synthesises the RNA primer

DNA polymerase III elongates the primer, until it bumps into the 5’ end of another primer and falls off the DNA

the DNA polymerase I removes the ribonucleotides at the 3’ end of the Okazaki fragment

finally the ligase joins the adjacent nucleotides between two fragments closing the single strand DNA break

Question 18

The trombone

Answer 18

both strands together

the two polymerases at the fork advance in the same direction

Question 19

What happens at the end of the DNA

Answer 19

in prokaryotes circular chromosomes, the last active DNA polymerase III will always encounter an RNA primer and DNA polymerase I and ligase will finish the job

but the linear eukaryotic chromosomes have a problem: the last RNA primer of the lagging strand cannot be made into DNA

Question 20

Telomeres

Answer 20

telomeres are special, repetitive sequences at the end of the eukaryotic chromosomal DNA

Stabilised by proteins

They can be extended after every cell cycle, if the cell has telomerase

They do not prevent but postpone after every cell cycle, if the cell has telomerase

They do not prevent but postpone the erosion of genes near the ends of DNA molecules as they provide a buffer

Question 21

Effects of telomeres on the germline

Answer 21

loss of telomeres would lead to offspring inheriting chromosomes lacking some genes

Question 22

Effects of telomeres on somatic cells

Answer 22

progressive loss of terminal genes would impact tissue renewal

Question 23

Effects of telomeres on medicine

Answer 23

cancer cells need to express telomerase and somatic cells lacking telomerase may be related to aging

Question 24

Cyclic activity of telomerase

Answer 24

elongation- reverse transcription (RNA to DNA)

translocation- RNA template ends have the same sequence

Question 25

Replication errors

Answer 25

minimised by proofreading capacity of enzymes but polymerase mistakes and DNA damage due to tension ahead of forks can cause changes in the sequence

Question 26

If there are errors in the germline:

Answer 26

sequence changes can pass to the next generation
depending on the phenotypic effect the changes may be maintained in the population
this allows evolution

Question 27

If there’s errors in somatic cells:

Answer 27

cells can become defective and be removed or accumulate as senescent cells decreasing performance of tissue

potential development of cancer (the incidence of tumours is related to the frequency of cell division )

Question 28

Mutagens

Answer 28

ionising radiation (X and gamma rays)

chemical

Question 29

Chemical mutagens

Answer 29

Alkylating agents: modify the bases in the DNA making the polymerases pair them with the wrong base during replication

nucleotide analogues: mimic the structure of a nucleotide to fool DNA polymerase to introduce them in DNA

intercalating agents: insert themselves between he nitrobases and can create frame shifts during replication

Question 30

DNA repair mechanisms

Answer 30

DNA polymerase proofread newly made DNA replacing incorrect nucleotides

in mismatch repair of DNA repair enzymes correct error in base pairing

in nucleotide excision repair, DNA is scanned for structural deformations, a nuclease cuts out and replaces damaged stretched of DNA and the gap is closed by polymerase/ligase activities