PMID- 10523676
OWN - NLM
STAT- MEDLINE
DCOM- 19991124
LR  - 20190508
IS  - 0270-7306 (Print)
IS  - 0270-7306 (Linking)
VI  - 19
IP  - 11
DP  - 1999 Nov
TI  - Molecular architecture of the mouse DNA polymerase alpha-primase complex.
PG  - 7886-96
AB  - 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.
FAU - Mizuno, T
AU  - Mizuno T
AD  - The Institute of Physical Research (RIKEN), Wako, Saitama 351-0198, Japan.
FAU - Yamagishi, K
AU  - Yamagishi K
FAU - Miyazawa, H
AU  - Miyazawa H
FAU - Hanaoka, F
AU  - Hanaoka F
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Mol Cell Biol
JT  - Molecular and cellular biology
JID - 8109087
RN  - 0 (Recombinant Proteins)
RN  - EC 2.7.7.- (DNA Polymerase I)
RN  - EC 2.7.7.- (DNA Primase)
RN  - EC 2.7.7.- (DNA polymerase alpha-primase)
SB  - IM
MH  - Animals
MH  - Binding Sites
MH  - COS Cells
MH  - DNA Polymerase I/chemistry/genetics/*metabolism
MH  - DNA Primase/chemistry/genetics/*metabolism
MH  - DNA Replication
MH  - Eukaryotic Cells
MH  - Mice
MH  - Models, Genetic
MH  - Models, Molecular
MH  - Molecular Biology/methods
MH  - Protein Binding
MH  - Protein Structure, Quaternary
MH  - Recombinant Proteins/chemistry/metabolism
MH  - Zinc Fingers
PMC - PMC84873
EDAT- 1999/10/19 00:00
MHDA- 1999/10/19 00:01
CRDT- 1999/10/19 00:00
PHST- 1999/10/19 00:00 [pubmed]
PHST- 1999/10/19 00:01 [medline]
PHST- 1999/10/19 00:00 [entrez]
AID - 10.1128/mcb.19.11.7886 [doi]
PST - ppublish
SO  - Mol Cell Biol. 1999 Nov;19(11):7886-96. doi: 10.1128/mcb.19.11.7886.