Cell Bio Exam 2 Lecture 2

Front 1

Give some examples of how double strand DNA breaks occur (5).

Back 1

Ionizing radiation (grays, Xrays)
Accidentally during replication
Meiotic recombination
V(D)J recombination for antibody assembly
Yeast mating type switching
Mobile genetic elements

Front 2

How do you find double strand DNA breaks if they occur at a random place in the genome?

Back 2

Neutral sucrose gradients and pulse field gels.

Front 3

How do you find double strand breaks if they occur synchronously, at predictable locations like during V(D)J recombination? What specific signal can you use and how does it work?

Back 3

Souther blotting and PCR

H2AX phosphorylation - this histone like protein is phosphorylated after a dsStrand break, causing the molecule to fluoresce

Front 4

T/F: Non-homologous end joining occurs in mammalian cells and Homologous recombination occurs in yeast.

Back 4

False! This is old dogma. It is now known that both yeast and mammalian cells prefer homologous recombination.

Front 5

T/F: NHEJ is a slow and error free process.

Back 5

False. The process if fast and error prone.

Front 6

What proteins are required for NHEJ? What is the catalytic subunit?

Back 6

DNA-Dependent protein kinase (made of Ku70/80, scid).

DNA Ligase IV

Front 7

What proteins are required for NHEJ?

Back 7

DNA ligase IV
DNA-dependent protein kinase: DNA-PK –> a complex of DNA-end binding Ku70/Ku80 and SCID, the catalytic subunit

Front 8

When does NHEJ occur?

Back 8

Commonly in cells where there is no homolog, like in haploid cells

Front 9

Give the overall NHEJ mechanism.

Back 9

Front 10

When is Rag1/Rag2 expressed?

Back 10

Only when immune cells are differentiating.

Front 11

What mechanism is NHEJ during V(D)J recombination similar to? Give a brief description of this process.

Back 11

Transposon insertion --> Cut, Circularize, recognize sequence, then fuse ends.

Front 12

T/F: NHEJ during Recombination is error free. Why is this important?

Back 12

False. Errors help increase genetic variability (diversity) in the development of the immune system.

Front 13

Why are translesion polymerases needed in Ab development?

Back 13

B/c the hypervariable reasons are subject to somatic hypermutations. Translesion polymerases are needed so RNA machinery can make proteins from this error filled DNA

Front 14

A DNA molecule experiences an accidental double strand break. Give the outcomes of this strand if A)Undergoes NHEJ or B)Undergoes Homologous recombination.

Back 14

A) Annealed but has missing nucleotides.

B) The sequence is completely stored by copying the second chromosome.

Front 15

What is ectopic recombination?

Back 15

A form of homologous recombination where the DNA is obtained from a different locus.

Front 16

What is interhomolog recombination?

Back 16

A form of homologous recombination where the DNA is obtained from the same locus on the homologous chromosome.

Front 17

What is sister chromosome recombination?

Back 17

A form of homologous recombination where the DNA is obtained from the same locus on a sister chromosome.

Front 18

What is site specific recombination?

Back 18

A type of ectopic homologous recombination where the DNA for the recombination is on an extrachromosomal element.

Front 19

What is homeologus recombination?

Back 19

A form of ectopic homologous recombination where the DNA sequence is similar, but not identical.

Front 20

What is unequal sister chromatid recombination?

Back 20

A form of ectopic homologous recombination where the sequence may be at a different locus on a sister chromatid.

Front 21

What type of ectopic homologous recombination results in loop out events?

Back 21

intrachromosomal recombination.

Front 22

T/F: Ectopic homologous recombination can occur intra and interchromosomally?

Back 22

True!

Front 23

Place the following steps of homologous recombination in order:

A) An exsposed 3' end pairs with its complement in the intact homolog. The other strand of the duplex is displaced.

B) The invading 3' end is extended by DNA polymerase plus branch migration, eventually generating a DNA molecule with two crossovers called Holliday intermediates.

C) Cleavage of the HOlliday intermediates by specialized nucleases generates either of the two recombination products. In product set 2, the DNA on either side of the region undergoing repair is recombined.

D) A double strand break in one of two homologs is converted to a double strand gap by the action of exonucleases. Strands with 3' ends are degraded less than those with 5' ends, producing 3' single strand extensions.

E) Further DNA replication replaces the DNA missing form the site of the original double-strand break.

Back 23

D A B E C

Front 24

In the strand resection step of homologous recombination, what enzyme creates the single stranded 3' end in e. coli? Eukaryotes?

Back 24

E. Coli = ReBCD helicase/nuclease.

Eukaryotes = MRN complex

Front 25

What is the structure of the MRN complex? What other molecule does this complex resemble? What is its fxnal subunit and what is that subunit's fxn?

Back 25

Should remind you of SMC proteins! Coil coil, globular structures. Complex of 3 proteins. MRE11 is both exo and endonuclease; this does the digestion.

Front 26

What two syndromes are associated with defects in the MRN complex?

Back 26

Niijmegen breakage syndrome (Nbs1)
Bloom’s syndrome (Sgs1)

Front 27

After limited digest by Mre11 subunit of the MRN complex during the resection step of homologous recombination, what proteins take over?

Back 27

After limited digest, Exo1 or
Sgs1 helicase/Dna2 take over

Front 28

What protein/DNA combo initiates synapsis (Eukaryotes and Prokaryotes)? Why is this step of homologous recombination important?

Back 28

A single strand of DNA wrapped by RecA (e.coli) or Rad51 (Eukaryotes).

This is the step where the DNA finds the perfect homolog...just a few bases in the WHOLE genome.

Front 29

Other than strand synapsis and exchange in homologous recombination, what function does RecA have?

Back 29

It has a regulatory fxn in the E. coli SOS system. It activates proteolyis of LexA, shutting down the system.

Front 30

What cofactor in the Homologous Recombination step: RecA mediated synapsis and strand exchange, is thought to be a marker for breast cancer? What molecule does this protein interact with?

Back 30

BRCA2

Rad 51

Front 31

What are the important cofactors in the Homologous Recombination step: RecA mediated synapsis and strand exchange? (E. coli and Eukaryotes)

Back 31

E.Coli: SSB

Eukaryotes: Replication factor A

Front 32

What is heteroduplex DNA - what protein mediates its formation and what is this type of DNA composed of?

Back 32

DNA that is a result of Strand Invasion mediated by Rec A. heteroduplex DNA is composed of strands from two different DNA helices.

Front 33

Heteroduplex DNA can have errors, what type of repair system does heteroduplex DNA serve as a substrate for?

Back 33

Mismatch repair.

Front 34

What can be a detrimental consequence of heteroduplex DNA?

Back 34

Gene conversion: You can lose an allele

Front 35

What is Gene Conversion? During what process does it occur?

Back 35

A process that occurs during the formation of heteroduplex DNA. It is a non-reciprocal transfer of Genetic information.

Front 36

After gene conversion, if two sequences are too unrelated, what does the mismatch repair system do? Why does it do this?

Back 36

It aborts the repair. This prevents interspecies crosses and genetic instability within the same organism.

Front 37

What are the structures found at Holliday junctions that cause branch migration?

Back 37

RuvA and RuvB

Front 38

What enzyme cuts holliday junctions to resolve them?

Back 38

Resolvase

Front 39

T/F: Resolvase cuts randomly at the holliday junction?

Back 39

True

Front 40

When resolvase cuts uncrossed strands, crossing over of flanking markers does or does not occur?

Back 40

Crossover of flanking markers does occur

Front 41

When resolvase cuts crossed strands, crossing over of flanking markers does or does not occur?

Back 41

Does not!