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Lecture PowerPoint to accompany Molecular Biology Fourth Edition Robert F. Weaver Chapter 22 Homologous Recombination Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 22.1 Homologous Recombination Pathways 22-2 RecBCD Pathway – Initial Binding RecBDC-sponsored homologous recombination in E. coli: – DNA helicase activity unwinds the DNA toward a Chi-site • Sequence 5’-GCTGGTGG-3’ • Chi sites found on average every 5000 bp in E. coli genome – RecBCD protein has • ds- and ss-exonuclease activity • ss-endonuclease activity • Activities permit RecBCD to produce a ss-tail now coated by RecA protein 22-3 The RecBCD Pathway Schematic RecBCD pathway is a well-studied homologous recombination pathway used by E. coli 22-4 RecBCD Pathway – D Loop • Invasion of a duplex DNA by a RecA-coated single-stranded DNA from another duplex that has suffered a double-stranded break • Invading strand forms a D loop (displacement) – Loop is defined by displaced DNA strand – When tail finds homologous region, nick occurs in in D-looped DNA – Nick allows RecA and ss-break create a new tail that can pair with gap in the other DNA • Subsequent degradation of the D-loop strand leads to the formation of a branched intermediate 22-5 Holliday Junctions • Branch migration in this intermediate yields a Holliday junction with 2 strands exchanging between homologous chromosomes • Branch in the Holliday junction can migrate in either direction by breaking old base pairs and forming new ones in a process called branch migration • This migration process does not occur at a useful rate spontaneously – DNA unwinding required – Unwinding requires helicase activity and energy from ATP 22-6 Resolving Holliday Junctions • Holliday junctions can be resolved by nicking 2 of its strands • Yielding: – 2 noncrossover recombinant DNAs with patches of heteroduplex – 2 crossover recombinant DNAs that have traded flanking DNA regions 22-7 22.2 Experimental Support for the RecBCD Pathway - RecA • • • The recA gene has been cloned and overexpressed with abundant RecA protein available for study It is a 38-kD protein that can promote a variety of strand exchange reactions There are 3 stages of participation of RecA in strand exchange 1. Presynapsis – RecA coats the ss-DNA 2. Synapsis – alignment of complementary sequences in ss- and ds-DNAs 3. Postsynapsis – ss-DNA replaces the (+) strand in ds-DNA to form a new double helix – Joint molecule is an intermediate in this process 22-8 Presynapsis In the presynapsis step of recombination: – RecA coats a ss-DNA participating in recombination – SSB accelerates the recombination process • Melting secondary structure • Preventing RecA from trapping any secondary structure that would inhibit strand exchange later in the recombination process 22-9 Synapsis: • Synapsis is the proper alignment of complementary sequences • Synapsis occurs when: – Single-stranded DNA finds a homologous region in a doublestranded DNA – This ss-DNA aligns with the ds-DNA • No intertwining of the 2 DNAs occurs at this point 22-10 Postsynapsis: Strand Exchange • RecA and ATP collaborate to promote strand exchange between ss- and ds-DNA • ATP is necessary to clear RecA off the synapsing DNAs • This makes way for formation of ds-DNA involving the single strand and one of the strands of the DNA duplex 22-11 RecBCD • RecBCD has a DNA endonuclease activity – Nicks ds-DNA especially near Chi sites – ATPase-driven DNA helicase activity that can unwind ds-DNA from their ends – The activities help RecBCD provide the ssDNA ends that RecA needs to initiate strand exchange 22-12 RuvA and RuvB • RuvA and RuvB form a DNA helicase that can drive branch migration • RuvA tetramer with square planar symmetry recognizes the center of a Holliday junction and binds to it • Likely induces the Holliday junction itself: – To adopt a square planar conformation – To promote binding of hexamer rings of RuvB to 2 diametrically opposed branches of the Holliday junction • RuvB uses its ATPase to drive the DNA unwinding and rewinding necessary for branch migration 22-13 A Synthetic Holliday Junction • Mix oligonucleotides at annealing conditions for complementary base-pairing • 5’-end of oligo 2 basepairs with the 3’-end of oligo 1 • 5’-end of oligo 1 basepairs with the 3’-end of oligo 2 • Ends cross over in pairing 22-14 RuvC • Resolution of Holliday junctions is catalyzed by the RuvC resolvase – This protein acts as a dimer to clip 2 DNA strands to yield either patch or splice recombinant products – Clipping occurs preferentially at the consensus sequence 5’-(A/T)TT(G/C)-3’ • Branch migration is essential for efficient resolution of Holliday junctions – Essential to reach preferred cutting sites – RuvA, B, and C work together in a complex to locate and cut those sites 22-15 Resolution of a Holliday Junction Holliday junction can be resolved in 2 ways: • Cuts 1 and 2 yield 2 duplex DNAs with patches of heteroduplex • Cuts 3 and 4 yield crossover recombinant molecules with the 2 parts joined by a staggered splice 22-16 22.3 Meiotic Recombination • Meiosis in most eukaryotes is accompanied by recombination • This process shares many characteristics with homologous recombination in bacteria • This section focuses on meiotic recombination in yeast 22-17 Mechanism Overview • Start with chromosomal lesion: ds-DNA break • Next exonuclease recognizes the break – Digests the 5’-end of the 2 strands – Creates 3’-single strand overhangs • One single-stranded end can invade other DNA duplex, forming a D loop • DNA repair synthesis fills in the gaps in the top duplex expanding the D loop • Branch migration can occur in both directions leading to 2 Holliday junctions • Holliday junctions can be resolved to yield either a noncrossover or a crossover recombinant 22-18 Model of Yeast Recombination 22-19 The Double-Stranded DNA Break • DNA cleavage uses 2 Spo11 – Active site Tyr as OH – Attack 2 DNA strands at offset positions – Transesterification reaction breaks phosphodiester bonds within DNA strands – Creates new bonds • Nicking DNA strands – Nicking is asymmetric – Yields 2 sizes oligos • Release of Spo11-linked oligos 12-37 nt long 22-20 DSB End Resection • Resection occurs on both strands using prior nicks • Recombinases load asymmetrically onto the newly created singlestranded regions • One protein tags coated free 3’-end for invasion into homologous duplex • This leads to initiating Holliday complex formation 22-21 Creation of Single-Stranded Ends at DSBs • Formation of the DSB in meiotic recombination is followed by 5’3’ exonuclease digestion of the 5’-ends at the break • Digestion yields overhanging 3’-ends that can invade another DNA duplex • Rad50 and Mre11 collaborate to carry out this reaction 22-22 22.4 Gene Conversion • When 2 similar, non-identical DNA sequences interact, possibility exists for gene conversion – Conversion of one DNA sequence into that of another • Sequences participating in gene conversions can be: – Alleles, as in meiosis – Nonallelic genes, such as the MAT genes that determine mating type in yeast 22-23 Gene Conversion Model • Strand exchange event with branch migration during sporulation has resolved to yield 2 duplex DNAs with patches of heteroduplex 22-24 Gene Conversion Without Mismatch Repair • Consider from the middle of the DSB recombination scheme • Invading strand is partially resected • DNA repair synthesis more extensive • Branch migration and resolution do not change nature of the 4 DNA strands 22-25