lecture 10- termination of replication

Question 1

describe termination of replication in eukaryotes

Answer 1

lagging strand synthesized in okazakis started by primers (adds 3’ OH)
- end of replication- primers removed from new DNA strans and replicated by dNTP’s
- when last primer removed, okazaki not fully copied each cell division, chromosome shortens - this is because polymerases cannot fill this gap without 3’ OH primer

Question 2

at the end of eukaryotic chromosomes are ___, causing an end replication problem which affects

Answer 2

telomeres
both chromosomes

Question 3

what is the function of telomeres

Answer 3

to protect ends of chromosomes from exonucleases (degradation) and DNA damage repair enzymes
- and maintain length of chromosomes

Question 4

who discovered telomeres, centromeres, and transposons?

Answer 4

Barbara McClintock

Question 5

describe Blackburn & Gall’s work with telomeres

Answer 5

studied ‘Tetrahymena thermophila’ because has 40,000 linear chromosomes (lots of telomeres)
- identified sequence of their telomeres: TTGGGG repeats
- studied other organisms- found telomere sequences to be simple tandem repeats (the number of repeats and the sequences fluctuates)
- humans have TTAGG repeats

Question 6

in vertebrates, including humans, telomere length ___ with age, but not in ___

Answer 6

declines
germ-line cells

Question 7

describe telomere shortening

Answer 7

every time cell replicates –> telomeres shorten
- after a number of replications, cell senescence (death)

Question 8

How can organisms survive after so many cell divisions (shortened telomeres)?

Answer 8

found that the enzyme telomerase can add telomere repeats on chromosomes, keeps size of chromosomes in equilibrium

Question 9

describe telomerase structure

Answer 9

Telomerase Reverse Transcriptase (TERT)- the catalytic core, carry a tightly bound, noncoding telomerase RNA (TR), can synthesize DNA from RNA template
- Telomerase: TERT-TR holoenzyme- 10 protein subunits and 451 RNA nucleotides
- the scaffold (TR) is the template

Question 10

telomerase is a ribonucleoprotein…

Answer 10

RNA & proteins
- RNA component is the template- 150-1300 nuc’s long
- protein component has catalytic activities:
. DNA polymerase- adds dNTP’s to 3’ end
. reverse transcriptase
. hybrid RNA/DNA helicase activity
- formation of hydrogen bonds makes this hybrid
- telomerase breaks the H bonds to dissociate
DNA/RNA hybrid

Question 11

describe the steps of how telomerase solves the “end replication” problem in eukaryotes

Answer 11

telomerase extends the 3’ end of the DNA template by adding dNTP’s (DNA polymerase) and is a reverse transcriptase (because presents RNA to serve as template)
- telomerase is very different from other DNA polymerases because it has its own template and it synthesizes ssDNA (other DNA poly’s synthesize dsDNA)
- after adding 6 nucleotide repeats, telomerase separates RNA/DNA hybrid and repositions on the telomere for extension on the next repeat
- as soon as telomerase extends 3’ ends, DNA-alpha comes to end of chromosome, synthesizes primer –> DNA polymerases able to convert telomerase-extended 3’ ssDNA terminus to DNA duplex

Question 12

At the end of telomerase function, still going to have____ but not as dramatic as it would be without ___

Answer 12

loss at end of chromosomes
telomerase

Question 13

when telomere ends are replenished by telomerase, what can continue?

Answer 13

cells can keep dividing

Question 14

in humans, what cells is telomerase active in?

Answer 14

active in stem cells & germ cells
- detectable in many normal adult cell types
- highly active in 90% of human tumors

Question 15

telomeres are highly associated with health and disease…

Answer 15

• shortening with aging, stress, smoking, alcohol, infection
• short telomeres = loss of protective structures, DNA damage response, limited replication, cell senescence

Question 16

name the negative side of activation of telomerase

Answer 16

increased incidence of tumors
- maintaining telomeres are essential to all cell growth
- inhibiting telomerase would theoretically kill immortal cancer cells

Question 17

name 2 ways telomeres are protected and regulated by proteins

Answer 17

1- telomere-binding proteins (shelterin)
2- telomere loop (t-loop)

Question 18

how do telomere binding proteins protect telomeres

Answer 18

• in mammals, telomere binding proteins called shelterin
• regulate telomerase activity, prevent telomeres from growing too long & protect chromosomes from joining (DNA repair) and exonucleases
• the 3’ terminus ssDNA is created by removal of primer, but it can be recognized by cells as bad, damaged DNA –> solution is protecting this G-overhang with shelterin, DNA poly enzymes then not able to access it

Question 19

how does the telomere loop (t-loop) protect telomeres

Answer 19

t-loop buries the 3’ end of the telomere, regulating its length
- DNA repair enzymes unable to access it
- if telomeres in open form (no shelterings of the 3’ overhang, the ends are accessible by telomerase