We have continued to exploit our monoclonal, anti-DNA polymerase alpha antibodies to provide new information about this central enzyme of chromosomal replication. First, using a permeable cell system, we have established the capacity of the neutralizing antibodies to inhibit replicative DNA synthesis in human cell nuclei. This shows the central role of polymerase alpha in chromosomal replication and provides a novel approach to studies of the role of this and other DNA polymerases in a variety of replicative and repair functions. Second, by using our immunoaffinity purification protocol, we have accumulated quantities of the purified polymerase holoenzyme and are now obtaining amino acid sequence data on several of the catalytically active peptides as a first approach toward gene isolation experiments. A third accomplishment has been the successful identification of a single genetic locus that is necessary and sufficient for the expression of human DNA polymerase alpha activity and localizes to the short arm of the human X chromosome, at the junction of bands Xp21.3-Xp22.1. By adapting this methodology, we can proceed to establish whether the coupled DNA primase activity is itself tightly linked to the identified genetic locus, and whether heterospecific subunit assemblies are detectable in the rodent-human somatic cell hybrid clones in the generation of DNA polymerase alpha holoenzyme complexes. Information will be provided from: 1) the ability of the nonneutralizing monoclonal antibodies to discriminate human and rodent polymerase alpha peptides; and 2) the ability of the neutralizing antibodies to cross-react with rodent polymerase alpha fractions, permitting independent isolation of the rodent and human holoenzyme species from the same rodent-human hybrid extracts. Fourth, we have developed an immumoprecipitation assay that permits identification of the polymerase alpha peptide after one hour of in vivo labelling of growing cells. The assay indicates that the primary translation product is the largest member of the large polypeptide family, i.e., a band of l80kDa; thus the smaller members of this polypeptide family likely result from specific degradative events as a consequence of purification. It is now possible to carry out physiological studies on this enzyme, e.g., time of biosynthesis in cell cycle, kinetics of translocation between cytoplasm and nucleus, kinetics of enzyme turnover, control of enzyme synthesis as cells pass between cycling and noncycling states, etc. A major goal will be the isolation of polymerase alpha/primase genetic sequences from human cDNA and genomic libraries. (I)

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Zhou, Yanjiao; Wang, Teresa S-F (2004) A coordinated temporal interplay of nucleosome reorganization factor, sister chromatin cohesion factor, and DNA polymerase alpha facilitates DNA replication. Mol Cell Biol 24:9568-79
Uchiyama, Masashi; Wang, Teresa S-F (2004) The B-subunit of DNA polymerase alpha-primase associates with the origin recognition complex for initiation of DNA replication. Mol Cell Biol 24:7419-34
Kai, Mihoko; Wang, Teresa S-F (2003) Checkpoint responses to replication stalling: inducing tolerance and preventing mutagenesis. Mutat Res 532:59-73
Gutierrez, Pedro J A; Wang, Teresa S-F (2003) Genomic instability induced by mutations in Saccharomyces cerevisiae POL1. Genetics 165:65-81
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Kelman, Z; Zuo, S; Arroyo, M P et al. (1999) The C-terminal region of Schizosaccaromyces pombe proliferating cell nuclear antigen is essential for DNA polymerase activity. Proc Natl Acad Sci U S A 96:9515-20
Liu, V F; Bhaumik, D; Wang, T S (1999) Mutator phenotype induced by aberrant replication. Mol Cell Biol 19:1126-35
Lee, S S; Dong, Q; Wang, T S et al. (1995) Interaction of herpes simplex virus 1 origin-binding protein with DNA polymerase alpha. Proc Natl Acad Sci U S A 92:7882-6
Dong, Q; Wang, T S (1995) Mutational studies of human DNA polymerase alpha. Lysine 950 in the third most conserved region of alpha-like DNA polymerases is involved in binding the deoxynucleoside triphosphate. J Biol Chem 270:21563-70

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