Molecular cloning and characterization of splice variants of human RAD50 gene

In this report, splice variants of human RAD50 (hRAD50) were cloned and cha racterized. A Northern blot survey identified two transcripts that hybridiz ed to a hRAD50 cDNA clone, an upper faint band (5.9 kb) and lower dense ban d (4.6 kb). cDNA clones (hRAD50-2, 4.6 kb) encompassing the entire hRAD50 t ranscript but having a shorter 3'-untranslated region (3'UTR) than the prev iously reported hRAD50-1 cDNA (5.9 kb; Dolganov, G.M., Maser, R.S., Novikov , A., Tosto, L., Chong, S., Bressan, D.A., Petrini, J.H.J., 1996. Human Rad 50 is physically associated with human Mre11: Identification of a conserved multiprotein complex implicated in recombinational DNA repair. Mel. Cell. Biol. 16, 4832-4841.) were isolated. The presence of AU-rich sequences in t he 3'UTR of hRAD50-1, which define mRNA instability and Northern results, s uggest that hRAD50-2 is the major transcript of hRAD50. A third alternative splice variant that lacks the ATP-binding domain was also identified (hRAD 50-3, similar to 4.5 kb). Expression of hRAD50-3 transcript was detected in all tissues examined by RT-PCR (reverse transcriptase-polymerase chain rea ction) and nested DNA-PCR analyses. Expression of hRAD50 partially rescued the MMS (methyl methanesulfonate)sensitive phenotype in rad50 mutant yeast, whereas hRAD50-3 did not show complementation. These data suggest that the hRAD50-3 does not repair DNA double-strand breaks most likely due to its i nability to bind ATP, and to bind damaged DNA. The existence of these alter native splice forms is potentially important in regulation of the biologica l activity of the DNA recombinational repair gene, hRAD50. (C) 1999 Elsevie r Science B.V. All rights reserved.