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.
We have isolated from a Lambda-gt 11 library a human cDNA clone with one open reading frame of about 2400 bases. A stretch of about 350 amino acids in the deduced amino acid sequence is up to 40 percent identical with parts of the known amino acid sequences of E. coli and yeast glutaminyl (Gln)-tRNA synthetase. The isolated cDNA sequence corresponds to an internal section of a 5500 bases long mRNA that codes for a 170 kDa polypeptide associated with Gln-tRNA synthetase. Thus, the human enzyme is about three times larger than the E. coli and two times larger than the yeast Gln-tRNA synthetase. The three enzymes share an evolutionarily conserved core but differ in amino acid sequences linked to the N-terminal and C-terminal side of the core.
Protein extracts were prepared at various times after serum stimulation of growth-arrested mouse 3T3 fibroblasts. The extracts were fractionated by sucrose gradient centrifugation and used to determine the activities of DNA polymerase tl and DNA primase. We found that polymerase and primase appeared in close association in one homogeneous 8.2-S peak. Neither polymerase, free of associated primase, nor primase, free of polymerase, could be detected at any time after serum stimulation. The activities of both enzymes started to increase concomitantly at the beginning of the DNA replication phase of the cell cycle. We found five to six times more DNA primase activity in replicating than in resting 3T3 cells.Besides DNA primase, a second additional priming activity could be detected. This activity sedimented at 12.5 S and corresponded most probably to RNA polymerase I.DNA primase initiates DNA synthesis by making short oligoribonucleotide primers, which are then elongated with DNA chains. Almost all nuclear mammalian DNA primases have been found in close association with DNA polymerase a. Isolated DNA polymerase tl consists of one large subunit of approximately 180 kDa and at least two smaller subunits of about 70 kDa and 55 kDa (recently reviewed in [l]). An apparently homogeneous DNA primase preparation, free of DNA polymerase, was isolated from mouse hybridoma cells. The hybridoma cell primase activity sedimented at 5.5 S and was associated with two polypeptides of 56 kDa and 46 kDa [2]. More recently a 5.5-S-primase-like activity could be recovered after treatment of purified mouse cell polymerase 01 with ethyleneglycol and dimethylsulfoxide [3, 41. The conclusion that DNA primase activity may be associated with one of the smaller polymerase subunits is consistent with reports describing a free DNA primase of 50-60 kDa from Drosophila melanogaster embryos [5] and yeast cells [6, 71. We would like to point out though that this conclusion has not remained unchallenged. Evidence has been presented suggesting that both functions, primase and polymerase, could be located on the large subunit of the enzyme [8,9]. In any case, since DNA primase is one of the key enzymes of the replication apparatus it is important to know how its activity is regulated. We have asked whether the enzymatic activity is expressed in a prereplicative phase or in the replicative phase (S phase) of the cell cycle. It has been well documented that the activity of DNA polymerase a increases with the beginning of the S phase (summarized in [l, 101); and recent evidence suggests that the activity increase is coupled to de novo synthesis of the large 180-kDa subunit. The biosynthesis of the smaller polymerase subunits is less strictly coupled to the S phase and could already be detected some time before the onset of DNA replication [lo].In the present work we have determined DNA primase and DNA polymerase activities at various times after mouse 3T3 fibroblasts had synchronously entered the cell cycle. OurCorrespondence to R. Knippers, Fakultat fur ...
We have used a cDNA encoding the core region of the human glutaminyl-tRNA synthetase to determine the chromosomal localization of the corresponding gene. Southern blots of restricted DNA from a panel of rodent-human cell lines and in situ chromosome hybridization gave identical results showing that the human gene locus for glutaminyl-tRNA synthetase resides on the distal long arm of chromosome 1. There are now nine mapped aminoacyl-tRNA synthetase genes in the human genome.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.