Testing predictions of the double-strand break repair model relating to crossing over in Mammalian cells.
In yeast, four-stranded, biparental "joint molecules" containing a pair of Holliday junctions are demonstrated intermediates in the repair of meiotic double-strand breaks (DSBs). Genetic and physical evidence suggests that when joint molecules are resolved by the cutting of each of the two Holliday junctions, crossover products result at least most of the time. The double-strand break repair (DSBR) model is currently accepted as a paradigm for acts of DSB repair that lead to crossing over. In this study, a well-defined mammalian gene-targeting assay was used to test predictions that the DSBR model makes about the frequency and position of hDNA in recombinants generated by crossing over. The DSBR model predicts that hDNA will frequently form on opposite sides of the DSB in the two homologous sequences undergoing recombination [half conversion (HC); 5:3, 5:3 segregation]. By examining the segregation patterns of poorly repairable small palindrome genetic markers, we show that this configuration of hDNA is rare. Instead, in a large number of recombinants, full conversion (FC) events in the direction of the unbroken chromosomal sequence (6:2 segregation) were observed on one side of the DSB. A conspicuous fraction of the unidirectional FC events was associated with normal 4:4 marker segregation on the other side of the DSB. In addition, a large number of recombinants displayed evidence of hDNA formation. In several, hDNA was symmetrical on one side of the DSB, suggesting that the two homologous regions undergoing recombination swapped single strands of the same polarity. These data are considered within the context of modified versions of the DSBR model.
Birmingham
Erin C
EC
Department of Molecular Biology and Genetics, College of Biological Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Lee
Shauna A
SA
McCulloch
Richard D
RD
Baker
Mark D
MD
D000818:Animals; D001483:Base Sequence; D003434:Crossing Over, Genetic / Q000502:physiology*; D004260:DNA Repair / Q000502:physiology*; D045566:DNA, Cruciform; D018390:Gene Targeting; D005819:Genetic Markers; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids / Q000502:physiology*
D045566:DNA, Cruciform; D005819:Genetic Markers