Properties of the nuclear P1 protein, a mammalian homologue of the yeast Mcm3 replication protein

Abstract

Polyclonal antibodies were raised against a multiprotein 'holoenzyme' form of calf thymus DNA polymerase alpha-primase and used to probe a human cDNA-protein expression library constructed in the lambda gt11 vector. The probe identified a series of cDNA clones derived from a 3.2 kb mRNA which encodes a novel 105 kDa polypeptide, the P1 protein. In intact cells, the P1 protein was specifically associated with the nucleus, and in cell extracts, it was associated with complex forms of DNA polymerase alpha-primase. The synthesis of human P1-specific mRNA was stimulated upon addition of fresh serum to growth-arrested cells, and RNA blot analyses with the human P1-cDNA probe indicated that P1 is encoded by a strictly conserved mammalian gene. The amino acid sequence deduced from a 240-codon open reading frame resident in the largest human P1-cDNA (0.84 kb) displayed greater than 96% identity with that deduced from the equivalent segment of a 795-codon open reading frame of a larger mouse P1-cDNA (2.8 kb). Throughout its length, the primary structure of mammalian P1 displayed strong homology with that of Mcm3, a 125 kDa yeast protein thought to be involved in the initiation of DNA replication (Gibson et al. 1990. Mol. Cell. Biol. 10: 5707-5720). The P1-Mcm3 homology, the strong conservation of P1 among mammals, its nuclear localization, and its association with the replication-specific DNA polymerase alpha strongly suggest an important role of the P1 protein in the replication of mammalian DNA.

Regulation of cell growth-dependent expression of mammalian CDC6 gene by the cell cycle transcription factor E2F

“…Overexpression of E2F could The mechanism of initiation of DNA replication in mammalian cells has been investigated by identifying counterparts homologous to yeast components involved in the regulation of initiation of DNA replication. Successful identi®cation of human homologs of ORC1 and 2, CDC6 and 7, and MCM2-7 (Fujita et al, 1996;Gavin et al, 1995;Hu et al, 1993;Musahl et al, 1995;Sato et al, 1997;Thommes et al, 1992;Todorov et al, 1995;Tsuruga et al, 1997b;Williams et al, 1997) suggests that the main stream of the regulatory mechanism involved in initiation of DNA replication is conserved between humans and yeasts. Indeed, the mechanisms that regulate the expression of CDC6 gene are similar in humans and yeasts.…”

Characterisation of a human homologue of a yeast cell division cycle gene, MCM6, located adjacent to the 5′ end of the lactase gene on chromosome 2q21

“…Several of these genes have been identified in vertebrate DNA and homologues have so far been identified for MCM2 (BM28 [8]), MCM3 (P1 [9]), MCM4 (CDC54/cdc21 [10]), MCM5 (CDC46 [10]), and MCM7 (cdc47 [10]). A human MCM6 homologue has not so far been reported.…”

Genomic structure of the gene for the human P1 protein (MCM3) and its exclusion as a candidate for autosomal recessive polycystic kidney disease

“…Putative exonintron boundaries were assigned by direct evaluation of the break points between the cDNA and the genomic sequences, based on the splicing consensus sequences ( Table 1). Given that the available cDNA sequence includes the entire coding region and is close in size (about 3.1 kb) to its message (about 3.2 kb), 7 our analysis most likely indicates that the MCM3 gene consists of 17 exons and 16 introns and spans at least some 20.7 kb of genomic sequence ( Figure 1).…”

“…X62153, consisting of a cDNA sequence of 2575 bp which included the entire proposed coding region. 7 Another cDNA was independently cloned later having been called human Replication Licensing Factor beta subunit gene (GenBank locus HUMHRLFB, accession No. D38073).…”

“…Its cDNA encodes a nuclear protein of 105 KDa and is 60% homologous to the yeast MCM3 (minichromosome maintenance deficiency) replication control gene of S. cerevisiae. 7 It is thought to be involved in the control of single DNAreplication per cell cycle in association with the replicationspecific DNA polymerase alpha. MCM3 is highly conserved in eukaryots and is ubiquitously expressed.…”