Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E DNA Repair Drosophila BLM in Double-Strand Break Repair
Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Bloom Syndrome: Men & Flys BLM is Bloom Syndrome Gene Predisposition to cancer BLM is a RecQ-type Helicase –Involved in synthesis dependent DS-break repair –Most double-strand break repair involves synthesis in flys (but not in plants, animals)
Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Synthesis Dep. Strand-annealing (SDSA) DS-break, processed to 3’ ss-DNA end Strand displacement and homolog annealing Prime DNA synthesis Gene conversion without crossing over Unlike other forms of DS-break repair Bloom Syndrome cells have increased cross-overs
Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Transposons make DS-breaks Used P-elements and copia elements to generate DS-breaks –P-element engineered to contain white gene (red) –Copia insertion (orange) creates apricot allele –Yellow in single copy, apricot in two copies –Conversion to red “measures” SDSA –Single LTR in intron – near wild-type expression
Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E
Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Interpreting Eye Colors... Effect of BLM mutation –Causes fewer red eyes, fewer apricot, more yellow –Less SDSA (38 fold less), more aberrant repair Why? Effect on synthesis during repair? –Little effect on fraction of extended ends, but... –Substantial effect on length of extended ends –Repair synthesis is significantly shorter (aborted?)
Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E
Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Helicases and Repair RecQ (E. coli) prevents association of microhomologies Or, helicase could just facilitate synthesis No effect on microhomology representation in repairs was observed Some NHEJ after aborted synthesis? Some templated additions as well Likely multiple pathways for aberrant repair
Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E