VL 21 (Michael Lenhard)

Question 1

Recombination

Answer 1

Homologous recombination is essential in meiosis for generating diversity and for chromosome segregation, and in mitosis to repair DNA damage and stalled replication forks.

Site-specific recombination involves specific DNA
sequences.

Somatic recombination – Recombination that occurs in nongerm cells (i.e., it does not occur during meiosis); most commonly used to refer to recombination in the immune system.

–> Recombination systems have been adapted for experimental use.

Picture 2
* heterozygous individual that inherited A, B on one chromosome and a, b on the other parental chromosome + no homologous recombination
→ no crossing over → gametes: AB, ab
* homologous recombination between non-sister chromatids→recombinant gametes
* every site that carries sequence homology is potentially a substrate for this recombination activity
* site-specific recombination is driven by sequence-specific recombinase enzymes
* somatic recombination refers to VDJ segments in immune systems during maturation of B/T cells

Question 2

Homologous recombination occurs between synapsed chromosomes in meiosis:

Answer 2

• Prophase 1: Chromosomes must synapse (pair) in order for chiasmata to form where crossing over occurs
• Meiosis stages can be correlated with molecular event at DNA level
• Chiasmata = crossing-over points between homologous chromosomes
• Leptotene: telomeres become bunched up
  →forming telomere bouquet
  →help homologous chromosomes to pair/find each other
• How is this process initiated? Initiation during leptotene (DSB initiate recombination)

Question 3

The Synthesis-Dependent Strand-Annealing Model

Answer 3

• synthesis-dependent strand-annealing (SDSA) model is relevant for mitotic recombination because in produces gene conversion from ds-breaks without associated crossovers
• difference: no second end capture with D loop; strands unwound; original invading strand is displaced
  →pairs up with 2nd 3 ́ overhanging end
  →result: no changes/mutations (happens only if process happens between sister- chromatids of same chromosome; non-sister chromatids
  →gene conversion)

Question 4

Double-Strand Breaks
Initiate Recombination

Answer 4

• 0th: DSB (e.g. from ionizing radiation/other damaging processes) introduced on chromosomes in meiotic cell
• 1st: 5 ́ ends of cut are shortened→3 ́ overhanging ends (bound by SSB)
• 2nd: invasion of 3 ́ overhanging end into duplex of non-sister chromatid
  →bp of 3 ́ end + intact non-sister
  chromatid
  →3 ́-OH group →DNA-Pol start →DNA-synthesis →strand extension →loop of ss-DNA of intact non-sister chromatid is extruded/displaced (→D loop)
• 3rd: D loop captures 2nd (top) 3 ́ overhanging end → DNA-synthesis + ligation → double holiday junction
• 4th: resolved double holiday junctions
• no cross-over end product: starts, ends with same color
• cross-over end product: start, ends with different color
• heteroduplex DNA:
  contains blue ss of top chromosome + red ss from red chromosome; can contain bp which don ́t fit together

Question 5

Tetrad analysis in Neurospora crassa – Gene conversion:

Answer 5

• Heteroduplex DNA + process that it leads to was identified by tetrad analysis
• Neurospora crassa: ascomycete; products of single miotic division are physically held together in ascus
• Postmeiotic mitotic division
  → spores formed + encapsulated; 8 instead of 4 meiotic products
• start: diploid individual, heterozygous for some recognizable marker that affects spore color (A: yellow, a: brown)
  →4:4 segregation
• in meiosis process: alleles have been changed
  →aberrant ratios
• spores A, a in 5:3 ratios must have arisen by one meitotic division from
  single haploid meiosis product
  →two daugthers with different genotypes
  →heteroduplex in one meiosis product a

Question 6

Yeast Can Switch Silent and Active Mating-Type Loci

Answer 6

• yeast mating-type locus MAT (mating type cassette) has either the MATa or MATα genotype
• dominant allele HO→switch mating type at frequency of 10-6
• allele at MAT = active cassette
• two silent cassettes: HMLα, HMRa
• switching occurs if MATa is replaced by HMRα/MATα is replaced by HMRa
• haploid cells
• fusion between cells of opposite mating types (MATa + MATα) → diploid cell → meiosis →4 haploid spores →novel allele combinations formed →increasing genetic diversity within population
• mating type controlled by mating type cassette
• active cassette + silent cassette (harbour information for a/α mating type)
• phenotypically expressed mating type is determined by active cassette
• mating type switch through gene conversion (information in silent cassette copied into active cassette→expressed)
• process uses silent cassette with opposite information than what is present in active cassette

Question 7

Unidrectional Gene Conversion is initiated by the recipient MAT Locus:

Answer 7

• Mating-type switching is initiated by ds break made at MAT locus by HO endonuclease
• Recombination event is a synthesis-dependent strand-annealing reaction
• HO endonuclease creates DSB at boundary of active mating-type cassette
  →strand of active mating-type cassette pairs with inactive neighbouring donor cassette
  →gene conversion
  →active MAT-information degraded
  →replacement through DNA-synthesis with information of donor silent cassette

Question 8

Model of meiotic homologous recombination:

Answer 8

• Spo11 = endonuclease which creates DSB
• somatic cells → non-crossover pathway
• rule of thumbs: most meiosis one chiasma per chromosome arm
• it matters how HJ is resolved → two crossover products or two non-crossover products

Question 9

Specialized Recombination involves specific sites:

Answer 9

specialized recombination involves reaction between specific sites that are not necessarily homologous
* recombinase:
E that catalyses site-specific recombination
* Phage lambda integrates into bacterial chromosome by recombination between the attP site on the phage + attB site on E. coli chromosome

• core sequence:
  DNA segment that is common to attachment sites on phage lambda, bacterial genomes
  –> location of recombination event that allows phage lambda to integrate
• phage is excised from chromosome by recombination between the sites at the end of linear prophage (attL + attR)
• phage lambda int encodes integrase→catalyses: integration reaction

Picture:
* requires specific DNA-sequences + sequence-specific recombinases

lysogenic cycle:
targeted integration →replicated
→ passed to daughter cells
* attP + attB with core sequences
* Integrase (Int; encoded by phage lambda) + integration host
factor (IHF; encoded by bacteria)
* Integration → result: prophage
* “bad times”: prophage excision

Question 10

Exploiting lambda integration for biotechnology

Answer 10

• Two DNAs (one of which carries attP sites <–> att B sites)
• BP reaction: causes recombination between attP + attB sites → recombinant DNA molecules with attL + attR sites; unidirectional
• LR reaction: different E mix; recognizes specific attachment sites; recombination

Question 11

Gateway Cloning

Answer 11

problem:
* vector combination with 250 TF genes of organism of interest → express proteins + two-hybrid screening

solution:
* integrate 250 cDNAs (encoding TFs) in 3 different vectors for 3 different applications
* gateway cloning: cDNAs have to be flanked by recognition sites
* destination vectors for expressing cDNA in E. coli + suicide gene
* transform resulting vectors in cells, where suicide gene kills them → expression clone

Question 12

Site-specific recombination resembles topoisomerase activity

Answer 12

• integrases related to topoisomerases
• recombination reaction resembles topoisomerase action except that nicked strands from different duplexes are sealed together
• reaction conserves energy by using a catalytic Tyr in the E to break a phosphodiester bond + link to broken 3 ́ end

Question 13

Recombination pathways adapted for experimental systems

Answer 13

• mitotic homologous recombination allows for targeted transformation
• Cre/lox + Flp/FRT systems allow for targeted recombination, gene knockout construction
• Flp/FRT system adapted to construct recyclable selectable markers for gene deletion

Cre/lox:
* Cre = site-specific recombinase; recognizes loxP
→Cre catalyses recombination between loxP-sites
* 2 different mouse-lines with Cre (+ promoter of interest (cell-type specific or inducible) + loxP-sites flanking one
exon of one GOI
* Cre-absence → nothing happens
* mouse crossing
* Cre-expressing cells: Cre binds two loxP-sites, catalyses recombination, cut out intervening sequence as circular DNA, reseal the rest as linear DNA, mutant allele or two loxP-flanked alleles and both recombined on other locus →homozygous mutant (gene function disrupted)
For which purpose might these conditional or cell-type specific knock-outs be particularly useful?
* study the post-embryonic function of essential genes

Flp/FRT (drosophila):
* Flp = recombinase
* FRT = recombination target site
* induced expression of Flp recombinase after chromosome
replication → Flp recognize, bind, catalyse recombination
between FRT-sites of non-sister chromatids
* mitosis → chromatids separated
* Flp/FRT system adapted to construct recyclable selectable
markers for gene deletion