Abstract
Bloom syndrome caused by inactivation of the Bloom DNA helicase (Blm) is characterized by increases in the level of sister chromatid exchange, homologous recombination (HR) associated with cross-over. It is therefore believed that Blm works as an anti-recombinase. Meanwhile, in Drosophila, DmBlm is required specifically to promote the synthesis-dependent strand anneal (SDSA), a type of HR not associating with cross-over. However, conservation of Blm function in SDSA through higher eukaryotes has been a matter of debate. Here, we demonstrate the function of Blm in SDSA type HR in chicken DT40 B lymphocyte line, where Ig gene conversion diversifies the immunoglobulin V gene through intragenic HR between diverged homologous segments. This reaction is initiated by the activation-induced cytidine deaminase enzyme-mediated uracil formation at the V gene, which in turn converts into abasic site, presumably leading to a single strand gap. Ig gene conversion frequency was drastically reduced in BLM(-/-) cells. In addition, BLM(-/-) cells used limited donor segments harboring higher identity compared with other segments in Ig gene conversion event, suggesting that Blm can promote HR between diverged sequences. To further understand the role of Blm in HR between diverged homologous sequences, we measured the frequency of gene targeting induced by an I-SceI-endonuclease-mediated double-strand break. BLM(-/-) cells showed a severer defect in the gene targeting frequency as the number of heterologous sequences increased at the double-strand break site. Conversely, the overexpression of Blm, even an ATPase-defective mutant, strongly stimulated gene targeting. In summary, Blm promotes HR between diverged sequences through a novel ATPase-independent mechanism.
Highlights
There are five human RecQ family proteins: Bloom DNA helicase (Blm), Wrn, RecQ1, RecQ4, and RecQ5
Analysis of Ig Gene Conversion in BLMϪ/Ϫ Cells—To investigate the role of Blm in synthesis-dependent strand anneal (SDSA), we examined the rate of Ig gene conversion
We measured the gain of surface IgM expression in subclones derived from wild type and BLMϪ/Ϫ cells that have a frameshift mutation in the IgV segment, as documented previously [25,26,27]
Summary
Plasmid Constructs—Gene targeting substrates were amplified with primers listed in supplemental Table S1. Analysis of Ig Gene Conversion—BLMϪ/Ϫ cells were established from CL18, a subclone of DT40 cells that is negative for sIgM [26]. The rate of Ig gene conversion was assessed by measuring the gain of sIgM expression as described previously [27]. Rearranged V was amplified by PCR with Pyrobest DNA polymerase (Takara Bio) (30 cycles of 94 °C for 30 s, 60 °C for 1 min, and 72 °C for 1 min) with 5Ј-CAGGAGCTCGCGGGGCCGTCACTGATTGCCG-3Ј and 5Ј-GCGCAAGCTTCCCCAGCCTGCCGCCAAGTCCAAG-3Ј primers, as described previously [31]. The targeting DNA fragment was amplified by PCR from the Mneo-1 to Mneo-4 substrate plasmid using Pyrobest DNA polymerase (Takara Bio) (30 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 2 min), with the 5Ј-GGATCGGCCATTGAACAAGATGGATTGCAC-3Ј and 5Ј-GGAAACAGCTATGACCATGATTACGCCAAG-3Ј primers. Measurement of Chromosome Aberrations—Chromosome aberrations were measured as described previously [32]
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