Molecular cloning and characterization of splice variants of human RAD50 gene.
In this report, splice variants of human RAD50 (hRAD50) were cloned and characterized. A Northern blot survey identified two transcripts that hybridized to a hRAD50 cDNA clone, an upper faint band (5.9kb) and lower dense band (4.6kb). cDNA clones (hRAD50-2, 4.6kb) encompassing the entire hRAD50 transcript but having a shorter 3'-untranslated region (3'UTR) than the previously reported hRAD50-1 cDNA (5.9kb; Dolganov, G.M., Maser, R.S., Novikov, A., Tosto, L., Chong, S., Bressan, D.A., Petrini, J.H.J., 1996. Human Rad50 is physically associated with human Mre11: Identification of a conserved multiprotein complex implicated in recombinational DNA repair. Mol. Cell. Biol. 16, 4832-4841.) were isolated. The presence of AU-rich sequences in the 3'UTR of hRAD50-1, which define mRNA instability and Northern results, suggest that hRAD50-2 is the major transcript of hRAD50. A third alternative splice variant that lacks the ATP-binding domain was also identified (hRAD50-3, approximately 4.5kb). Expression of hRAD50-3 transcript was detected in all tissues examined by RT-PCR (reverse transcriptase-polymerase chain reaction) 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 inability to bind ATP, and to bind damaged DNA. The existence of these alternative splice forms is potentially important in regulation of the biological activity of the DNA recombinational repair gene, hRAD50.
Kim
K K
KK
Research Institute of Medical Sciences, Chonnam University, 5 Hakdong Dongku, Kwangju 501-190, South Korea. kimkk@chonnam.chonnam.ac.kr
Shin
B A
BA
Seo
K H
KH
Kim
P N
PN
Koh
J T
JT
Kim
J H
JH
Park
B R
BR
D020413:3' Untranslated Regions / Q000235:genetics; D017766:Acid Anhydride Hydrolases; D000255:Adenosine Triphosphate / Q000378:metabolism; D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics; D005816:Genetic Complementation Test; D006801:Humans; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D015003:Yeasts / Q000187:drug effects / Q000235:genetics / Q000254:growth & development
D020413:3' Untranslated Regions; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C086930:RAD50 protein, S cerevisiae; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D008741:Methyl Methanesulfonate; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D045643:DNA Repair Enzymes