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Molecular and Cellular Biology, November 1999, p. 7886-7896, Vol. 19, No. 11
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Molecular Architecture of the Mouse DNA Polymerase alpha -Primase Complex

Takeshi Mizuno,1 Kumiko Yamagishi,1 Hiroshi Miyazawa,1,2 and Fumio Hanaoka1,3,*

The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198,1 National Institute of Public Health, Meguro, Tokyo 108-8638,2 and Institute for Molecular and Cellular Biology, Osaka University, Suita, Osaka 565-0871,3 Japan

Received 24 May 1999/Returned for modification 28 June 1999/Accepted 9 August 1999

The DNA polymerase alpha -primase complex is the only enzyme that provides RNA-DNA primers for chromosomal DNA replication in eukaryotes. Mouse DNA polymerase alpha  has been shown to consist of four subunits, p180, p68, p54, and p46. To characterize the domain structures and subunit requirements for the assembly of the complex, we constructed eukaryotic polycistronic cDNA expression plasmids expressing pairwise the four subunits of DNA polymerase alpha . In addition, the constructs contained an internal ribosome entry site derived from poliovirus. The constructs were transfected in different combinations with vectors expressing single subunits to allow the simultaneous expression of three or four of the subunits in cultured mammalian cells. We demonstrate that the carboxyl-terminal region of p180 (residues 1235 to 1465) is essential for its interaction with both p68 and p54-p46 by immunohistochemical analysis and coprecipitation studies with antibodies. Mutations in the putative zinc fingers present in the carboxyl terminus of p180 abolished the interaction with p68 completely, although the mutants were still capable of interacting with p54-p46. Furthermore, the amino-terminal region (residues 1 to 329) and the carboxyl-terminal region (residues 1280 to 1465) were revealed to be dispensable for DNA polymerase activity. Thus, we can divide the p180 subunit into three domains. The first is the amino-terminal domain (residues 1 to 329), which is dispensable for both polymerase activity and subunit assembly. The second is the minimal core domain (residues 330 to 1279), required for polymerase activity. The third is the carboxyl-terminal domain (residues 1280 to 1465), which is dispensable for polymerase activity but required for the interaction with the other three subunits. Taken together, these results allow us to propose the first structural model for the DNA polymerase alpha -primase complex in terms of subunit assembly, domain structure, and stepwise formation at the cellular level.

* Corresponding author. Mailing address: Institute for Molecular and Cellular Biology, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-7975. Fax: 81-6-6877-9382. E-mail: fhanaoka{at}imcb.osaka-u.ac.jp.

Molecular and Cellular Biology, November 1999, p. 7886-7896, Vol. 19, No. 11
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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