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Carcinoma of the cervix is one of the most common malignancies. Papanicolaou (Pap) smear tests have reduced mortality by up to 70%. Nevertheless their interpretation is notoriously difficult with high false-negative rates and frequently fatal consequences. We have addressed this problem by using affinity-purified antibodies against human proteins that regulate DNA replication, namely Cdc6 and Mcm5. These antibodies were applied to sections and smears of normal and diseased uterine cervix by using immunoperoxidase or immunofluorescence to detect abnormal precursor malignant cells. Antibodies against Cdc6 and Mcm5 stain abnormal cells in cervical smears and sections with remarkably high specificity and sensitivity. Proliferation markers Ki-67 and proliferating cell nuclear antigen are much less effective. The majority of abnormal precursor malignant cells are stained in both low-grade and high-grade squamous intraepithelial lesions. Immunostaining of cervical smears can be combined with the conventional Pap stain so that all the morphological information from the conventional method is conserved. Thus antibodies against proteins that regulate DNA replication can reduce the high false-negative rate of the Pap smear test and may facilitate mass automated screening.

A minichromosome maintenance (MCM) protein complex has been implicated in restricting DNA replication to once per cell cycle in Xenopus egg extracts, based on the behavior of a single protein, XMCM3. Using a two-hybrid screen with XMCM3, we have identified a novel member of the MCM family in Xenopus that is essential for DNA replication. The protein shows strong homology to Saccharomyces cerevisiae MCM7 (CDC47) and has thus been named XMCM7. XMCM7 is present in a multiprotein complex with other MCM proteins. It binds to chromatin and is displaced from chromatin by the act of replication. XMCM7 does not preferentially colocalize with sites of DNA replication but colocalizes with XMCM3 throughout replication. Immunodepletion of the MCM complex from Xenopus egg extract by anti-XMCM7 antibodies inhibits DNA replication of sperm and permeable HeLa G2 nuclei but not permeable HeLa G1 nuclei. Replication capacity of the Xenopus egg extract immunodepleted of the MCM complex by anti-XMCM7 antibody can be rescued by MCM proteins eluted from anti-XMCM3 antibody. We conclude that both proteins are present in the same complex in Xenopus egg extract throughout the cell cycle, that they remain together after binding to chromatin and during DNA replication, and that they perform similar functions.