Ribosomal Protein S3 Negatively Regulates Unwinding Activity of RecQ-like Helicase 4 through Their Physical Interaction

Abstract

Edited by Patrick SungHuman RecQ-like helicase 4 (RECQL4) plays crucial roles in replication initiation and DNA repair; however, the contextual regulation of its unwinding activity is not fully described. Mutations in RECQL4 have been linked to three diseases including Rothmund-Thomson syndrome, which is characterized by osteoskeletal deformities, photosensitivity, and increased osteosarcoma susceptibility. Understanding regulation of RECQL4 helicase activity by interaction partners will allow deciphering its role as an enzyme and a signaling cofactor in different cellular contexts. We became interested in studying the interaction of RECQL4 with ribosomal protein S3 (RPS3) because previous studies have shown that RPS3 activity is sometimes associated with phenotypes mimicking those of mutated RECQL4. RPS3 is a small ribosomal protein that also has extraribosomal functions, including apurnic-apyrimidinic endonuclease-like activity suggested to be important during DNA repair. Here, we report a functional and physical interaction between RPS3 and RECQL4 and show that this interaction may be enhanced during cellular stress. We show that RPS3 inhibits ATPase, DNA binding, and helicase activities of RECQL4 through their direct interaction. Further domain analysis shows that N-terminal 1-320 amino acids of RECQL4 directly interact with the C-terminal 94 -244 amino acids of RPS3 (C-RPS3). Biochemical analysis of C-RPS3 revealed that it comprises a standalone apurnic-apyrimidinic endonuclease-like domain. We used U2OS cells to show that oxidative stress and UV exposure could enhance the interaction between nuclear RPS3 and RECQL4. Regulation of RECQL4 biochemical activities by RPS3 along with nuclear interaction during UV and oxidative stress may serve to modulate active DNA repair.The RecQ class of helicases function at various stages of DNA metabolism. The presence of at least one homolog in each species evidences their crucial role in maintaining genomic stability. In humans, three of five RecQ helicases have been associated to different genetic disorders. WRN and BLM have been linked with Werner's syndrome and Bloom's syndrome, respectively, and RECQL4 has been linked with Rothmund-Thomson syndrome (RTS), 2 Baller-Gerald syndrome, and RAPADILINO syndrome. Of these, RECQL4 is unique on the basis of its domain structure and function. The N terminus of this protein represents the only human homolog of Saccharomyces cerevisiae essential replication initiation protein Sld2 (1, 2). The helicase domain is conserved across the RecQ class of helicases. Initial reports suggested RECQL4 lacked of helicase activity (3), but annealing, strand exchange and helicase activities of RECQL4 were later demonstrated (4 -7). Human RECQL4 was shown to unwind up to 22-bp double-stranded regions through its helicase activity (6, 7). ATPase and annealing activities of human RECQL4 have also been extensively studied (7,8). Robust helicase and annealing activities on substrates of various lengths have been reported for Drosophila melanogaster R...

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