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Molecular Cloning of CDK7-associated Human MAT1, a Cyclin-dependent Kinase-activating Kinase (CAK) Assembly Factor¹

Department of Biochemistry and Biophysics [A. Y., Y. X.] and Curriculum in Molecular Biology and Genetics [M. A. N., Y. X.], University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3280; Division of Orthopaedic Surgery, Research Institute of Childrens Hospital Los Angeles, Los Angeles, California 90027 [L. W., F. L. H.]; Department of Molecular Pharmacology and Toxicology, University of Southern California School of Pharmacy, Los Angeles, California 90033 [F. L. H.]; and Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 [R. K.]

Mammalian CDK7 is a protein kinase identified as the catalytic subunit of cyclin-dependent kinase (CDK)-activating kinase and as an essential component of the transcription factor TFIIH that is involved in transcription initiation and DNA repair. We have identified in human cells a number of CDK7-associated cellular proteins that appear to fall into two classes based on their relative [³⁵S] metabolic labeling intensity. One class of proteins present in CDK7 immunocomplexes as a minor fraction contains components of the TFIIH transcription complex such as p62 and p89^ERCC3, whereas the other fraction contains four polypeptides (p35, p37^{Cyclin H}, p75, and p95) that are stoichiometrically associated with CDK7. Whereas the levels of association of p35, p37^{Cyclin H}, and p75 with CDK7 remain unchanged between density-arrested and proliferating Ewing sarcoma EW-1 cells, the association of p95 with CDK7 was significantly decreased as cells reached confluency. Through a large-scale immuno-purification of CDK7 complexes and protein microsequencing, we have isolated a cDNA that encodes p35 and have shown that it is the human homologue of Mat1 that is involved in the assembly of CAK. MAT1 contains a highly conserved C₃HC₄ motif at its NH₂ terminus, a characteristic feature shared among RING finger proteins. The human MAT1 gene expresses a single 1.6-kb transcript, the steady-state level of which, like CDK7 and cyclin H, varies significantly in different cell lines and in different terminally differentiated tissues.

¹ This study was supported by NIH Grant CA 65572 to Y. X. Y. X. is a recipient of an American Cancer Society Junior Faculty Award and is a Pew Scholar in Biomedical Science. The sequence reported in this paper has been deposited in GenBank under accession no. X92669. M. A. N. is supported by a grant from the Lucille P. Markey Foundation to the program in Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill.

² To whom requests for reprints should be addressed, at 215 Fordham Hall, Campus Box 3280, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280.

Received 11/13/95. Accepted 11/16/95.

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