Molecular Biology Fourth Edition 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

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

The RecBCD Pathway Schematic RecBCD pathway is a well-studied homologous recombination pathway used by E. coli

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

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

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

22.2 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 Presynapsis – RecA coats the ss-DNA Synapsis – alignment of complementary sequences in ss- and ds-DNAs Postsynapsis – ss-DNA replaces the (+) strand in ds-DNA to form a new double helix Joint molecule is an intermediate in this process

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

Synapsis: Synapsis is the proper alignment of complementary sequences Synapsis occurs when: Single-stranded DNA finds a homologous region in a double-stranded DNA This ss-DNA aligns with the ds-DNA No intertwining of the 2 DNAs occurs at this point

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

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 ss-DNA ends that RecA needs to initiate strand exchange

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

A Synthetic Holliday Junction Mix oligonucleotides at annealing conditions for complementary base-pairing 5’-end of oligo 2 base-pairs with the 3’-end of oligo 1 5’-end of oligo 1 base-pairs with the 3’-end of oligo 2 Ends cross over in pairing

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

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

22.3 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

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

Model of Yeast Recombination

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

DSB End Resection Resection occurs on both strands using prior nicks Recombinases load asymmetrically onto the newly created single-stranded regions One protein tags coated free 3’-end for invasion into homologous duplex This leads to initiating Holliday complex formation

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

22.4 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

Gene Conversion Model Strand exchange event with branch migration during sporulation has resolved to yield 2 duplex DNAs with patches of heteroduplex

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