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Cloning, overexpression, and genomic mapping of the 14-kDa subunit of human replication protein A
C B Umbricht1, L F Erdile, E W Jabs
1Department of Molecular Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Abstract:
Replication protein A (RPA) is a three-subunit protein that plays a central role in eukaryotic DNA replication, recombination, and repair. We have previously reported the cloning and bacterial expression of the 70- and 32-kDa subunits of human RPA (hRPA). We have now cloned the 14-kDa subunit (hRPA3) from a HeLa cell cDNA library. The hRPA3 cDNA is a 692-base pair sequence that contains an open reading frame encoding a protein of 121 amino acids with a calculated molecular mass of 13.6 kDa. The deduced amino acid sequence shows only limited similarity to the small subunit of yeast RPA and is unrelated to any other protein in the current data banks. A recombinant protein containing a short histidine tag at the NH2 terminus has been purified in good yield from Escherichia coli by metal-chelate affinity chromatography. Antibodies prepared against recombinant hRPA3 recognize the native protein and inhibit SV40 DNA replication in vitro. We have localized the genes for the 70-, 32-, and 14-kDa subunits to chromosomes 17, 1, and 7, respectively, using polymerase chain reaction amplification of genomic DNA from rodent-human hybrid cell lines. Since RPA appears to be involved in several fundamental cellular processes, the physical mapping of the RPA genes may be useful in identifying possible human genetic defects associated with RPA deficiency or dysfunction.
Insights
Researchers cloned the 14-kDa subunit of human Replication Protein A (hRPA3), crucial for DNA repair. This finding aids in understanding genetic defects related to RPA dysfunction.
Area of Science:
- Molecular Biology
- Genetics
Background:
- Replication protein A (RPA) is essential for DNA replication, recombination, and repair in eukaryotes.
- Previous work established the cloning and expression of the 70- and 32-kDa subunits of human RPA (hRPA).
Purpose of the Study:
- To clone and characterize the 14-kDa subunit of human RPA (hRPA3).
- To investigate the function and chromosomal localization of hRPA3.
Main Methods:
- Cloning hRPA3 from a HeLa cell cDNA library.
- Expressing and purifying recombinant hRPA3 in E. coli using metal-chelate affinity chromatography.
- Generating antibodies against recombinant hRPA3 to detect native protein and assess functional inhibition.
- Mapping RPA subunit genes to specific chromosomes using PCR on rodent-human hybrid cell lines.
Main Results:
- The hRPA3 cDNA sequence was determined, encoding a 121-amino acid protein (13.6 kDa) with limited similarity to yeast RPA.
- Recombinant hRPA3 was successfully purified and antibodies recognized the native protein, inhibiting SV40 DNA replication in vitro.
- Genes for hRPA subunits (70, 32, and 14 kDa) were mapped to chromosomes 17, 1, and 7, respectively.
Conclusions:
- The characterization of hRPA3 provides a complete set of human RPA subunits for further study.
- Gene localization of RPA subunits is valuable for investigating genetic disorders linked to RPA deficiency or dysfunction.