Oct 28

Question 1

How does Spo11 catalyze DSB formation? When done?

Answer 1

It has a Tyr, the OH acts as nuclophile for transesterification. Each of them acts on a backbone, so get a ds break. Done naturally for meiotic recombination

Question 2

Once 2 Spo11 molecules cleave the 2 phosphate backbones, how does recombination happen after?

Answer 2

1. The Mre11-Rad50-Xrs2 complex binds to each Spo 11, acts as nuclease and cuts on 5’ end of it, so taking off 5’ off one strand leaves a 3’ overhand on the other.
2. Sae2 degrades the 5’ ends a bit more, even bigger overhand
3. Sgs-Dna2/Exo1 complex degrades 5’ ends more, get very long 3’ overhangs.
4. RPA and RecA recombinases attach to 3’ overhands for recombination

Question 3

What is gene conversion and what are the 2 outcomes of it?

Answer 3

It is non-reciprocal transfer of genetic info. It can result in non-crossover or crossover. Either way 2 homologous chromosomes end up switching DNA

Question 4

When does recombination occur during mitosis?

Answer 4

It is rarer than meiosis, but it can occur wiht ionizing radiation, and ds breaks. It happenss in S and G2 phases of the cycle (before actual mitosis) as it goes G1, S, G2, mitosis. When DSB occurs it halts cell cycle for recombination to occur

Question 5

How is mitotic DSB repaired?

Answer 5

1.Get 3’ overhands and RPA stabilization just like meiosis
2.Next what is diff is Rad 52, sometimes BRCA2 recruited, now get Rad 51 too. Then go via the Synthesis dependent strand annealing pathway or the double strand break repair pathway

Question 6

Which Rad does which invasion of strands?

Answer 6

Rad 51 does 1st invasion, 52 does second

Question 7

When is yeast haploid or diploid? What are teh mating types?

Answer 7

It is diploid unless if conditions not good, then haploid. It can be a or alpha

Question 8

Where is genetic info stored and expressed in S. cerevisiae (yeast)?

Answer 8

As haploid, MATalpha info is stored, not expressed in HMLalpha. MATa info is stored, not exressed in MMRa. Then if gene put in MAT locus then it is expressed?

Question 9

What are the steps for recombinational DNA repair of DSB in yeast?

Answer 9

1. The HO nuclease makes DSB at MAT locus
   2.THe free DNA ends are given 3’ overhang
   3.Rad 51 binds to overhands, directs strand invasion
2. The original mating type info removed with nuclase digestion
3. DNA pol extends invading 3’ end
   6.

Question 10

How get nonhomologous end joining? When in cell cycle, what is phosphorylated etc.

Answer 10

1. In G1 or G0, Ku70 and 80 recruited to broken ends, then DNA-PK and Artemis bind, hold together broken ends
2. The helicase separates DNA
   3.The kinase activity of DNA-PK activates endonuclease activity of Artemis, get cleavage of DNA, get 3’ sticky ends.
3. The strands are annealed together, then gaps filled in with DNA pol
4. Nicks sealed with XRCC4 and DNA ligase IV. Some removal after breakage so not perfect as before, have some mutations

Question 11

How track location of protein using CRISPR?

Answer 11

You can add green fluorsent protein tag to DNA on N or C terminus

Question 12

What is other words for template strand?

Answer 12

Non-coding and anti-sense strand

Question 13

What is DNA dependent RNA synthesis?

Answer 13

This is RNA transcription

Question 14

How many RNA pol eukary vs prokary

Answer 14

Prokary has 1, eukary have 3

Question 15

Transcription of DNA into RNA is both ___ and ___

Answer 15

Multiple transcription of same gene at same time and translation can occur rapidly as RNA transcript dissaciates from DA template a few ribonulcotides behind point of synthesis

Question 16

Does RNA transcription have same proofreading as DNA replication?

Answer 16

Has much less proofreading

Question 17

What each of the Eukary DNA pol for?

Answer 17

Pol 1:Ribosomal RNA transcription
2: protein encoding genes
3. tRNA and smaller rRNA transcritpion