Lecture 6 - DNA Repliaton, Regulation & Repair

Question 1

making a replication machine at an origin:

Answer 1

A DNA binding protein complex that can initiate the assembly of the replication machinery initiator) binds to the origin (DnaA in bacteria, Orc/Cdc6 in eukaryotes)

The initiator prepares the origin for the first step of replisome assembly, promoting some unwinding in prokaryotes and is necessary for recruitment of the helicase of the replisome onto the DNA

Question 2

initiation in bacteria:

Answer 2

•The bacterial chromosome has a single start site (“oriC”) for DNA replication

•Binding the initiator protein DnaA triggers the process

Question 3

when is DNA replication began in bacteria?

Answer 3

•At least 10 proteins are involved in the start of DNA synthesis in bacteria
•DNA methylation controls the timing. After replication, another round will not start until the new strand (of oriC) is methylated
•This in turn is triggered by as yet unknown processes in the plasma membrane (which binds the oriC region of the chromosome)

Question 4

DNA replication in the eukaryotic cell cycle compared to prokaryotic:

Answer 4

•Bacteria can divide continuously with DNA replication restarting before division is completed
•Eukaryotes have complex cell cycles in which DNA replication is a critical control step and ensures that DNA is only replicated once per cell cycle.

Question 5

eukaryotic initiation:

Answer 5

• multiple proteins bind to origins of replication
• sequence specificity varies widely in eukaryotes, some organisms such as yeast have a consensus sequences, humans don’t but frequently have the same chromatin modifications

Question 6

how do Orc proteins and CDC6 do a similar job to dnaA?

Answer 6

through binding to the origin and recruiting replisome proteins

Question 7

what do cyclin proteins do in eukaryotic initiation?

Answer 7

Cyclin proteins “licence” initiation and ensure each origin is only used once per cell cycle in part by phosphorylating ORC proteins.

Question 8

what can be found in the telomere sequences?

Answer 8

multiple end-to-tail repeats of a short TG-rich sequence TTAGGG

Question 9

although most of the repeats in telomere ends are double stranded…

Answer 9

… the 3’ end extends beyond the 5’ as a single strand

Question 10

how can the 3’ end in telomeres be extended?

Answer 10

this 3’ end can be extended further by telomerase, an enzyme containing a short RNA molecule complementary to the TTAGGG repeats

This RNA acts as a template for “reverse transcription”, copying the RNA into DNA (and thus extending the 3’strand of the telomere)

Question 11

how is the 5’ strand extended?

Answer 11

in the usual way, with an RNA primer and DNA polymerase

Question 12

how do telomeres differ with age?

Answer 12

Telomeres in young people are longer than those in older people

Question 13

how does telomerase change as we age?

Answer 13

adult somatic cells lose their telomerase activity

Question 14

what cells retain telomerase activity?

Answer 14

stem cells and germ cells retain telomerase activity

Question 15

once cancer cells start multiplying they have to…

Answer 15

activate telomerase to escape rapid senescence

Question 16

DNA damage and proof-reading:

Answer 16

even with proof reading, DNA polymerases make too many errors (about 100-1000 per cell division in humans)

many mutagens (chemicals, UV etc) continuously damage DNA - generating thousands of lesions per cell per day

Question 17

dangers of DNA damage:

Answer 17

•Cancer is caused by somatic cell mutations

•Cell mutations build up over your lifetime

•Many diseases are due to faulty DNA repair mechanisms which cause early onset of pathologies due to a high mutation burden

Question 18

what does persistent DNA damage lead to?

Answer 18

Persistent DNA damage leads to chromosome breakage and rearrangements which are readily visible to images of metaphase chromosome stained with chromosome paints

Question 19

Primary non-replicative causes of DNA damage are:

Answer 19

alklyation
depurination
deamination
UV irradiation

Question 20

Damaged DNA is fixed by bespoke repair pathways:

Answer 20

MMR – mismatch repair
UVER – UVDE mediated excision repair
NER – nucleotide excision repair
MGMT - O(6)-Methylguanine-DNA-methyltransferase mediated repair
BER – Base excision repair
HR – homologous recombination
NHEJ – nonhomologous end joining

Question 21

DNA repair mechanisms:

Answer 21

•Base-excision repair
•Nucleotide-excision repair
•Double strand break repair

Question 22

Base-excision repair: why U AND T?

Answer 22

•Deamination of C to U is a common damage to DNA

•In ancient cells where U was likely the only base that could base pair with A, DNA repair enzymes would not have been able to distinguish “real” from “damaged” U

•Replacing U with T means that all U in DNA must be damage and therefore repaired by base excision repair.

Question 23

Nucleotide excision repair:

Answer 23

•Important for removing lesions such as thymidine dimers that distort the double helix

•A substantial length of one strand is removed and replaced

•Critical to survival

Question 24

one of the earliest markers of uncontrolled cell growth:

Answer 24

accumulation of broken chromosomes

Question 25

DNA lesions can be bypassed by specific DNA polymerases:

Answer 25

replication starts at lesion

bypass DNA polymerase binds to lesion

bypass polymerase makes DNA opposite to the lesion.

replication starts

Question 26

integrity of DNA:

Answer 26

• DNA replication musty be rapid and accurate
• high fidelity DNA synthesis is not enough, repair mechanisms are also essential
• Despite the sophisticated machinery that evolution has produced, errors still accumulate. That’s likely why (even without disease or predators) we eventually die.

Question 27

accumulating errors:

Answer 27

DNA replication error rate = c. 1 in 1010
•So: Every two cell divisions there will, on average, be ≥1 error in DNA replication
•Generating an adult human requires about 45-50 cell divisions (246 = 7 x 1013), starting from the fertilised egg
•So: on average an adult human cell contains many new mutations acquired since fertilisation

Question 28

good news and bad news about DNA damage and repair:

Answer 28

good news:
•If 95% of DNA is “junk”, most mutations won’t be in coding regions (but 1-2 still will be; and some “junk” matters)
•Even in coding regions, many mutations don’t change protein function
•We have two copies of each gene

bad news:
•There will actually be far more than 45-50 cell divisions over a lifetime, because some cells divide continuously (skin etc)
•Mutations don’t just arise from DNA replication errors (chemicals, UV etc)

Question 29

end replication problem leads to:

Answer 29

gradual loss of telomere sequences. The combination of telomere repeats and telomerase action ensure that telomeres are not eroded

Question 30

DNA damage and repair is essential to…

Answer 30

health over a lifetime

Question 31

Both single strand and double strand repair pathways of are required to…

Answer 31

maintain genome integrity

Question 32

Both prokaryotic and eukaryotic cells define which DNA sequences will act as…

Answer 32

origins of DNA replication by using an initiator protein complex to recruit the helicase component of the replisome, but the details of the pathways differ.