VL 21 (Michael Lenhard) Flashcards
Recombination
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
Homologous recombination occurs between synapsed chromosomes in meiosis:
- 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)
The Synthesis-Dependent Strand-Annealing Model
- 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)
Double-Strand Breaks
Initiate Recombination
- 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
Tetrad analysis in Neurospora crassa – Gene conversion:
- 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
Yeast Can Switch Silent and Active Mating-Type Loci
- 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
Unidrectional Gene Conversion is initiated by the recipient MAT Locus:
- 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
Model of meiotic homologous recombination:
- 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
Specialized Recombination involves specific sites:
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
Exploiting lambda integration for biotechnology
- 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
Gateway Cloning
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
Site-specific recombination resembles topoisomerase activity
- 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
Recombination pathways adapted for experimental systems
- 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
