Molecular and Cellular Biology, November 1999, p. 7886-7896, Vol. 19, No. 11
The Institute of Physical and Chemical
Research (RIKEN),
Received 24 May 1999/Returned for modification 28 June
1999/Accepted 9 August 1999
The DNA polymerase
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Copyright © 1999, American Society for Microbiology. All rights reserved.
Molecular Architecture of the Mouse DNA Polymerase
-Primase Complex
-primase complex is the only enzyme that
provides RNA-DNA primers for chromosomal DNA replication in eukaryotes. Mouse DNA polymerase
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
. 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
-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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