A DNA polymerase-{alpha}{middle dot}primase cofactor with homology to replication protein A-32 regulates DNA replication in mammalian cells.
alpha-Accessory factor (AAF) stimulates the activity of DNA polymerase-alpha.primase, the only enzyme known to initiate DNA replication in eukaryotic cells ( Goulian, M., Heard, C. J., and Grimm, S. L. (1990) J. Biol. Chem. 265, 13221-13230 ). We purified the AAF heterodimer composed of 44- and 132-kDa subunits from cultured cells and identified full-length cDNA clones using amino acid sequences from internal peptides. AAF-132 demonstrated no homologies to known proteins; AAF-44, however, is evolutionarily related to the 32-kDa subunit of replication protein A (RPA-32) and contains an oligonucleotide/oligosaccharide-binding (OB) fold domain similar to the OB fold domains of RPA involved in single-stranded DNA binding. Epitope-tagged versions of AAF-44 and -132 formed a complex in intact cells, and purified recombinant AAF-44 bound to single-stranded DNA and stimulated DNA primase activity only in the presence of AAF-132. Mutations in conserved residues within the OB fold of AAF-44 reduced DNA binding activity of the AAF-44.AAF-132 complex. Immunofluorescence staining of AAF-44 and AAF-132 in S phase-enriched HeLa cells demonstrated punctate nuclear staining, and AAF co-localized with proliferating cell nuclear antigen, a marker for replication foci containing DNA polymerase-alpha.primase and RPA. Small interfering RNA-mediated depletion of AAF-44 in tumor cell lines inhibited [methyl-(3)H]thymidine uptake into DNA but did not affect cell viability. We conclude that AAF shares structural and functional similarities with RPA-32 and regulates DNA replication, consistent with its ability to increase polymerase-alpha.primase template affinity and stimulate both DNA primase and polymerase-alpha activities in vitro.
Casteel
Darren E
DE
Department of Medicine and Cancer Center of the University of California, San Diego, La Jolla, California 92093, USA.
Zhuang
Shunhui
S
Zeng
Ying
Y
Perrino
Fred W
FW
Boss
Gerry R
GR
Goulian
Mehran
M
Pilz
Renate B
RB
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D019915:DNA Primase / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D007939:Leukemia L1210 / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D014176:Protein Biosynthesis; D055503:Protein Multimerization; D011506:Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014162:Transfection
D011506:Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051716:Replication Protein A; D019915:DNA Primase; C116932:DNA polymerase alpha-primase; D004256:DNA Polymerase I