DNA Replication meets Genetic Exchange… Jacqueline Jonuschies

Replication Fork  Connecting points between two different mechanisms. 1. DNA-Replication 2. Homologous Recombination

Recombination vs. Replication  DNA-Replication:  Identical copies of chromosomes  Synthesize complement strand  Homologous Recombination  New genetic combinations, different alleles  Reestablish original pairing

History 1966Hanawalt: replication fork collapse (nicks) 1972 Strauß: breakage of stalled replication fork 1974 Skalka: repair of replication fork via homologous recombination

Replication fork falters, when:  it encounters an unrepaired DNA lesion (ss interruption in template)  Replication fork collapse  Generating ds-end  Chromosome fragmentation

 Progress blocked by a DNA-bound protein (stalled replication fork):  Form a Holliday junction  DNA-duplex  Exonucleolytic degradation of duplex or  Branch migration Overcome block Replication fork falters, when:

On the next slide we look at the structure of the DNA at this junction

 DNA junction-processing enzymes break replication fork  Chromosome fragmentation, when replication fork process is inhibited  Can be prevented by inactivation of Holliday junction-processing enzymes Replication fork falters, when:

Repair and Restoration  Repair via homologous recombination  Invasion of ds-end in sister duplex  Holliday junction  Replication fork restored

Homologous Recombination in T4  Early DNA replication: initiated by ori  Late DNA replication: primed from recombination intermediates System:  Replication substrate = circular plasmid  During T4 infection: ori – replication stops  plasmid and T4 share homology: replication resumed

Plasmid Replication by T4  Result: Concatemers (rolling circle)  Replication products of T4 replisomes: hydroxylmethyl cytosines  defend from restriction enzymes  Distinguish from unreplicated DNA

T4 Repair  DSB between partially homologous plasmids induced  T4 infection …  Homology-dependent end-invasion  Unidirectional ->T4 primes bidirectional replication

T4 Repair  Ss interruption:  ssDNA-binding protein: gp32 prevent binding of helicases + recombinase  UvsY (recombination-mediator protein): - disrupt gp32 cooperativity - allow UvsX (recombinase) to bind ssDNA  UvsX: invasion of ssDNA into homologous duplex  Gp32 binds displaced strand

 RAD: resistance to ionization radiation  Ionization: DSB -> RAD for repair 2 pathways RAD51/54/55/57RAD50/58/59/60 interhomolog intersister Yeast RAD52

Recombination for DNA repair  rad 52: essential for recombination! (Single-strand annealing, Double-strand invasion) Homologous recombination is more a DNA-repair mechanism than generating genetic diversity!!!

Repair mechanisms in yeast  rad27 (flap endonuclease)  pol30 (DNA clamp)  Rfc 1 (DNA clamp loader)  cdc9 (DNA ligase) mutants show defect in Okazaki fragment maturation accumulate ss-interruptions

Repair mechanisms in yeast  Combined with inactivated DSB-repair mutants -> lethal Ss-interruptions converted into DSB

Gene mutaions in yeast  RPA (ss-binding protein)  pol  : deficient in 3’->5’ exonuclease  Dna2p (helicase, nuclease) defect in Okazaki-fragment maturation strongly dependent on RAD50/51/52 Ss nick -> Replication fork collapse -> DSB

Recombinational Repair  Rad 51: in foci in S-Phase cells  functions in DNA repair (Organizer, catalyst)  Double Strand Repair: 1. ds-end processed by Rad50/58/60 -> ss- overhang -> RPA binding 2. Rad52: replacement of RPA->Rad51 3. Rad51 (Rad54/55/57): invasion of ds-end into intact sister duplex

Single Strand Annealing  Rad52-promoted annealing:  No homology to recA/rad51 recombinase  Bind ssDNA (form oligomeric rings/7mer)  36nt (ss-end) around heptamer signal purposes nucleation site for rad51/ RPA strand annealing reaction, block homologous recombination

Conditions for SSA  Regions of ssDNA at replication fork ( always such regions present)  Rate of DNA synthesis is slow ( eucaryotes)  Rad52 reattach end to chromosome

Central reaction of recombinational repair is not Rad51-catalyzed ds-end invasion!!! DSB-repair SSA

Duplex Opening  Rad54 promoted (DNA helicase)  Enhances insertion of ssDNA into cccDNA  Rad54: unwinds region of duplex  Interaction with Rad51 (bound to DNA)  Homology search simpler  Generate strand complementary to ss-end  Ss-annealing (rad52) -> rRNA

Homologous End-Joining  DSB: no recombination with sister/homolog…  Misalignment (microhomology)  Rad50/58(mre11)/60(xrs2)  Rad58: unwinds, degrade, anneal DNA strands  Catalyze strand assimilation  Limited hydrolysis of 5’end  Anneal 2 ss-tails at microhomologous regions Rad50/Mre11/Nbs1 -> higher eucaryotes