The putative nuclear localization signal of the human RAD52 protein is a potential sumoylation site.
RAD52, a key factor in homologous recombination (HR), plays important roles in both RAD51-dependent and -independent HR pathways. Several studies have suggested a link between the functional regulation of RAD52 and the protein modification by a small ubiquitin-like modifier (SUMO). However, the molecular mechanism underlying the regulation of RAD52 by SUMO is unknown. To begin investigating this mechanism, we identified possible target sites for sumoylation in the human RAD52 protein by preparing a RAD52-SUMO complex using an established Escherichia coli sumoylation system. Mass spectrometry and amino acid sequencing of the enzymatically digested fragments of the purified complex revealed that the putative nuclear localization signal located near the C terminus of RAD52 was sumoylated. Biochemical studies of the RAD52-SUMO complex suggested that sumoylation at the identified site has no apparent effect on the DNA binding, D-loop formation, ssDNA annealing and RAD51-binding activities of RAD52. On the other hand, visualization of the GFP-fused RAD52 protein in the human cell that contained mutations at the identified sumoylation sites showed clear differences in the cytosolic and nuclear distributions of the protein. These results suggest the possibility of sumoylation playing an important role in the nuclear transport of RAD52.
Saito
Kengo
K
Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.
Kagawa
Wataru
W
Suzuki
Takehiro
T
Suzuki
Hidekazu
H
Yokoyama
Shigeyuki
S
Saitoh
Hisato
H
Tashiro
Satoshi
S
Dohmae
Naoshi
N
Kurumizaka
Hitoshi
H
D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D051721:Rad52 DNA Repair and Recombination Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011995:Recombination, Genetic; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry / Q000378:metabolism*
D004268:DNA-Binding Proteins; D019913:Nuclear Localization Signals; D010446:Peptide Fragments; C448305:RAD51C protein, human; C497194:RAD52 protein, human; D051721:Rad52 DNA Repair and Recombination Protein; D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins