1.

Homologus recombination
2. DNA base pairing
1. pairing reactions require extensive regions of homology
2. need productive regions of homology
3. repairing a rep fork
1. topioisomerase is causing nicks
2. if there is a nick that is not repaired
3. movement of the replication strand leads to a double strand break
4. an exonuclease is recruited in and it will degrade the 5' end of one of the strands of DNA
5. creates a single strand
1. required in all type of HR
2. 3' overhang
6. strand invastion
1. unique feature of HR
2. invade into the ds (newly forming sister chromatid)
3. invades because this region in the ds is homolougous to the single strand
7. what opens up the dsDNA to allow HR
8. DNA replication is occuring on the leading strand
9. block to replication is overcome
10. topioismeroase usually fixes its own nick, this is what happens when it doesnt
4. RecA
1. Rad51 in eukaryotes
2. for strand invasion cannot have an intact DNA helix
1. can invad but wont base pair
5. RecA/Rad 51
1. picture
1. RecA hold together the invading strand and the DNA
1. get heteroduplex: one strand of DNA from one helix, and one from another helix
6. next slide
1. homology: to the region its interacting with
2. Rad52: when have exonuclease chew back to creat 3' overhang, the ssbs straighten it out
1. ssbs have to be replaced by Rad52
2. Rad52 allows for RecA binding
7. Branch Migration
1. picture
2. spontaneous: bottom strand is original, invading is above, branch point is when the pink
goes in and displaces the green
1. because there are regions of homology the pink strand as much likelihood of interacting
with template as green because as homologous
2. can move in either direction
3. no net change in nucleotides in the double helix
8. homolous recombination requires a sister chromatid
1. Cell cycle phase: S or G phase
2. 5' end is degraded for 3' overhang by exo nuclease
3. strand invasion: need ssb then Rad52 then RecA
4. DNA replication to extend branch point further
9. this is an example
1. pairing of newly synthesized with top strnad
2. dna ligation
 3. DNA rep to repair the top strand gap where the double helix broken
10. sexual
1. meiosis occurs
2. in gametes
11. why important
1. During meiosis have controlled form of HR
2. homologs recognize by unknown mechanisms
1. during pairing the homologs exchange DNA
3. creates diverstiy
12. Focusing
1. homolougous pairs
2. during meiosis, exchange of DNA material between pairs
3. blue: sites of gene vonbersation
4. red: sign of crossng over
1. does not occur when HR used to repair DNA
1. only when mieosis
13. Mitosis vs Meiosis
1. a round of rep then 2 rounds of cell division
14. prophase I
1. cromosomal condensation
2. recomnination
3. chiasm
15. The synaponemal complex
16. steps to generation
1. synapsis which is K to HR
17. synaptomal complex
18. mammalial proteins
1. mjaor proteins
19. the integrity
1. immunofoursecence, looking at a protein expressed by a cell
2. SCP3 and Dap1 (nuclear stain)
3. come in with an aB to the protein
4. have a tag at the end of the aB it corresponds to where the antigen is
5. lets say we start with a rabbit aB
6. come in with a different aB that is recognizing a rabbit aB and that has the visualizing tag
7. scp3 expressed but not localized then forms clumps around nucleus
8. in zygotene it forms long strips where it has localized along the axial element of the
chromosome
9. Pachetyne: clear alignment if indiv chromosomes
1. bound and synapsed so they look like one! Looks ½ the amount as zygotene
10. diplotene
1. see resoltuion of chiasmata at the end
2. HR in packaging
20. generatiing a target vector
1. target the scp2 locus
2. targetting from exon 1 to 6
3. contains NeoR
4. and has toxin has negative selection
5. KO exon 1 2 and 3
 6. letters below are restriction enzymes sites
21. SCP3 mutant cells
1. infertility
2. look at germ cells and found the phenotype are in the mitotic cells
3. take single cells and stain with silver nitrate which detects protein
4. scp3 is essential for the synaptonemal complex
1. the highly proteinated structure in cells is missing
22. scp3 mutant cells
1. can look at cempA aB or auto antibodies
2. B is packaging stage: full synapsis
3. C is contromeres only → so scp3 so its a null mutant and no product, missing the complex
4. dots in B and in C lots of dots, which indicates not close pairing of chromosomes
23. SCP1
24. SCP1 mutant cells
1. look at chromosomes in the miotic cells and see if there is a central core
1. axial elements are dark
2. in mutant the axial is fine but the central core is missing
2. right is using immunflourescense
1. K: leptotene
1. scp3 protein is unaffected
2. wt looks the same as mutant
3. bottom two panels are prophase
1. Q: fully snypased
2. R: mutants; the homologs are closed, and axial lateral elements forming BUT not fully
synapsed
25. SCP2 function in mice
1. A: wild type locus
1. targeting 39-43 locus disrupted
2. 1-38 in tact
2. B: can tell the difference by sizes restriction enzyme digest
3. C: western blot: wt, hetero, mutant
1. bottom is loading controlled
2. truncated form in -/-
1. truncated protein
26. SCP2 mechanisms
1. crest stains for centromeres
2. A: in packatine
3. C: silver nitrate staining; forming normally
4. B: show no effect of generation of scp3
1. but scp3 is not localized correctly
2. truncated form of scp2 is affecting the localization of scp3 (which is wildtpye)
1. sufficient to disrupt localization to lateral filament
27. truncated scp2 still binds to scp1
1. A: zygotene
2. scp1 can still be recruited to pairing chromosomes and azial is partially assembled
3. vast majority of chromsomes not synapsing correctly
28. Synapsis
1. formation of branch point
29. not randomly breaking DNA in HR in meiosis: programmed
30. the programmed ouble strand break
1. its a endonuclease
31. Spo11
1. paired due to scp1/2/3
2. spo1 recruits Mre11
3. Spo11 cuts one
1. dissociate
4. Mre is the exonuclease responsible for chewing to leave 3' overhand
5. one difference is the expression of spo11
32. why do
1. northern blots
2. probed for spo11 RNA
3. only one organ in body: testis: gametes: meiosis
4. only in meoitic cells in males
5. ovary meiosis only in fetal time
6. but in males all the time
33. title
34. spo11 mutants arrest
1. RAD51 is not correctly localized
2. scp3 is ok, but stuck in zygotene
1. but formation of A/L fine
3. if no rad51
1. then no HR; specifically no strand invasion
35.
1.
2.