BIO 431 Exam 1: End of DNA Replication and Telomeres (lecture 6)

• Ori

• bidirectionally

• Terminator

Tus; helicases

• Topoisomerase II

Termination in Prokaryotes​:

• ___ are at definedlocations​

• DNA Replication occurs ​_____ in E. coli​

• ____ sequencesare at defined locations​

Circular gene... so its different than linear chromosomes in euk ​

An area where termination occurs... just like there is an origin of replication

Continued:

​_____ proteins bind to terminator sequences and inhibit ____ ​

Replication fork can pass through theTus-DNA complex in one direction but not the other​

• Starts at the top... replication fork goes both ways ​

• ******Termination sequences (in orange) bind certain proteins (tus) to keep it going only one way (like a one way street) - can only unwind so far and meet in the middle at the bottom ​

• ****Not ALL end at the bottom... sometimes the termination sequence is 75% through the circle... just depends on where it is ​

Cont:

• ______ makes double-stranded DNA breaks​

• **separates the physical linkage of linked DNA circular molecules ​

The genomes are linked together ​

Use topiosmerase 2 gets them apart and reseals them so they are separated ​

origins of replication

Termination inEukaryotes​

Multiple ______ that come together and expand until everything is ruplicated

But.. What happens at the ends of linear chromosomes? ​​

1. Replication

2. repair

degraded

3' end of lagging strand

DNA sequence

Telomeres:

2 Issues​

1. End of ____ problem​

2. DNA ____ problem​

telomeres are at the end of chromosomes ​

Yellow is telomeres ​

Blue is chromosomes ​

Telomeres solve two different issues – the end of replication and DNA repair problem

Problem: the last RNA primer used on the strand is ____ and nothing is there to replace it ​

So, there is a small piece of DNA that is not replicated (on ______) ​

Everytime that DNA is duplicated... you lose a little bit of _____

• 300

13

Telomeres were discovered ​in Tetrahymena:

• Tetrahymena contains many(~___) very short linear chromosomes ​

• Elizabeth Blackburn discovered that the chromosomal ends had repetitive sequences​

• Did this result suggest that therepetitive sequence protectedthe ends of chromosomes? ​

Tetrahymena has 300 linear small chromosomes ​

Repetitive sequences at the end of chromosomes ___ bp long ​

*****Suggests that replicative sequences protect the chromosome (bc why else would you have the same sequence at the ends of 300 chromosomes)

• degraded

• repetitive sequences

Telomeres Protect ​Chromosome ends​:

• Tried to create manufactred chromosomes... but everytime they did they got _____ ​

• Did a test to discover if the ________ protected the chromosomes ​

• Add sequence to artificial chromosomes ​

• *****They did not degrade! They were maintained! ​

• 10,000-15,000

• genes

6; GGGTTA

telomere

DNA replication ​Problem Solved​:

Telomeres are repetitive sequences: In humans the repeat unit is GGGTTA. ​

• A telomere can be long—measured telomeres in humans are 10,000-15,000 nucleotides in length.​

• A telomere does NOT contain ___​

Suggests that the end of the chromsomes the repetitve units protect the chromosomes ​

Humans have ___ bp that are repeated (can be repated 10,000-15,000 bp – 2,500 6-bp repeats)​

Does not contain any genes... just repeats! ​

When RNA primer gets replaced... you lose some DNA... but you are losing the ____ not your actual genes! ​

PH buffer: resists changes in pH... telomeres are kind of like that! Main purpose is to be lost to save the actual coded chromosome

Telomerase

telomere

maintain/elongate

Discovery of telomerase​:

Elizabeth Blackburn and her graduate student, Carol Greider tested “cell extracts” from tetrahymena and discovered a fraction that had the ability to maintain/elongate telomeres! ​

​

_____ – enzyme ​

_____ – DNA sequence ​

Extracts that can ______ telomeres ​

What was different: had enzyme telomerase – able to maintain telomeres ​

Somatic

Stem cells

Somatic

Stem

RNA and protein

• end

• bottom

• 5'-3'

Two different types of cells in the body: somatic cells and stem cells ​

____ – regular body cells; limited divisions​

_____ – can live and divide forever; they are also undifferentiated ​

Most body cells are differeniated and somatic (limited in divisions) ​

Also have stem cells that can divide forever! WHY???? ​

___ cells have little or no telomerase – means that telmoeres shorten (why we age!!) ​

___ cells have telomerase – means that the telmoere length is stable ​

​

Telomerase is made up of _____​

Blue – piece of RNA that binds to DNA making a RNA-DNA hybrid

In order to lengthen... ​

• Binds at very ___ of telmoeric sequence (green is protein/enzyme) that adds bases​

• Not back filling... it extends it little by little ​

• ****After its extended... an RNA primer is laid down and the DNA pol jumps in and synthesizes the ____ strand... RNA is degraded again and process repeats ​

• Can ONLY be lengethened bc of telomerase ​

• ANY pol is going ___​​

****Telomerase is usually not in somatic cells... but cancer cells turn it on so it grows out of control and will grow forever ​

Read and comprehend

Telomere length control in yeast:

• Always gravitated towards a certain length ​

• Wanted to mainatin a certain length ​

double

mutations

telomeric

DNA repair problem​:

• Appreciated by Hermann Muller and Barbara McClintock inthe 1930’s: termed the word “telomere”​

​

• Creation of the new ends led to chromosomal arrangements (chromosomesfusing to each other)​

• Part of DNA repair pathways because of double-stranded breaks. ​

• Chromosomal arrangements led to aneuploidy​

• Natural ends of chromosomes are protected from chromosomal rearrangements​

• Why did natural ends not look like double-stranded breaks? ​

Second issue that it helps with: DNA repair problem ​

Knew radiation was bad for DNA – causes ____ stranded breaks ​

There are mechanisms in place to put them back together... but can cause ____ ​

Chromosome rearrangements... end result is often cancer ​

So, cells need to be able to correct these as needed ​

How do the cells know the difference between the two ends?? ​

Only fuse back together the ends that do NOT have ____ sequences! This helps differentiate what is an outside break vs an inside break in the middle of the sequence ​

​

• T-Loop​

proteins

DNA repair problem​:

• Solution is a ____

• The 3’protruding end has shown to loop back and “tuck” into the telomeric sequence of DNA duplex. ​

• Protects it from degradative enzymes and distinguishes them from broken DNA​

​

3' overhang... all repetitive sequences​

• Means that they are all the same! ​

This DNA loops out and fold back.. Displacing some of the front part... creating a loop! ​

End result: a loop that maks its base pairs ​

Can do this because it’s the same sequence! Its just repeats! ​

Ends are hidden in this loop (like a barbell... one loop for each end of the chromosome) ​

Lots of ____ bind to telemoric sequence that also help hide the ends as well ​

This makes the ends of chromosomes look different than an internal break

Senescence

dividing

______:

• telomere length gradually decreases with cel divisions to a point where descendent cells will inherit defective chromosomes and stop dividing

• shown in fibroblasts

• involved with cancer and aging

In most of our cells (somatic) we lose telomeres everytime ​

When our cellshit a certain lengeth of telomeres... there is a protection mechanism that stops it from ____ (so they don't die! Just stops dividing) ​

Somatic cell telomeres shorten over time​:

• Graph that shows telomere length at birth and as you age ​

• Decreases as you get older ​

• More to immortality than just telomerase being active! ​

1. important DNA

2. Tumor supressors get mutated... so senescene does not occur

3. progresses; dividing​

4. tumor; diversity

Telomere Shortening in Aging cells​:

1. Why would it be advantageous to activate tumor suppressor pathways that promote cell senescence when telomeres get critically short?​

• Telomere goes from long to short ​

• Activates tumor suppressor pathways that promote senecence so that they do not get rid of ______

2. Can you think of mutations that might result in cells becoming cancerous with critically short telomeres?​

3. What would happen if cancer cells reactivate telomerase? ​

• No longer has telomeric DNA... so now its just a broken DNA that does not loop ​

• Now creates chromosomal fusions... that create unstable changes and can even possibly change chromosomal numbers... this is MESSY and BAD ​

• Cell fuses them together... causes problems! ​

• Cancer cells reactivate telomerase... which means cancer _____ and keeps ____ ​

​4. Evolutionary reason for the telomereshortening in somatic cells: ​

• To suppress ___ growth. ​

• To ensure a ifinite lifespan for genetic ___​