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The primary structure of the 32-kDa subunit of human replication protein A

L F Erdile1, M S Wold, T J Kelly

  • 1Department of Molecular Biology and Genetics, Johns Hopkins Medical School, Baltimore, Maryland 21205.

The Journal of Biological Chemistry
|February 25, 1990
PubMed

Insights

Researchers isolated a cDNA for the 32-kDa subunit of Replication Protein A (RP-A), a key complex for simian virus 40 DNA replication. This discovery enables further study of RP-A

Area of Science:

  • Molecular Biology
  • Virology
  • Biochemistry

Background:

  • Replication Protein A (RP-A) is a heterotrimeric complex essential for DNA replication.
  • RP-A is composed of 70, 32, and 14 kDa subunits.
  • Simian virus 40 (SV40) DNA replication in vitro absolutely requires RP-A.

Purpose of the Study:

  • To isolate and characterize the cDNA encoding the 32-kDa subunit of RP-A.
  • To enable the production of the 32-kDa subunit for further functional studies.
  • To investigate the role of the 32-kDa subunit in SV40 DNA replication.

Main Methods:

  • Construction of an oligonucleotide probe based on tryptic peptide sequence.
  • Screening of a lambda gt11 HeLa cDNA library.
  • Overexpression of the cloned cDNA in bacteria using an inducible T7 expression system.
  • Generation of monoclonal antibodies against RP-A subunits.

Main Results:

  • Isolation of a cDNA encoding the 32-kDa subunit of RP-A.
  • Predicted amino acid sequence confirmed tryptic peptide sequences and molecular mass.
  • No significant homology found in GenBank database.
  • Bacterial expression produced a protein recognized by a specific monoclonal antibody.
  • Monoclonal antibody against the 32-kDa subunit inhibited in vitro DNA replication.

Conclusions:

  • The isolated cDNA successfully encodes the 32-kDa subunit of RP-A.
  • The 32-kDa subunit plays a critical role in SV40 DNA replication, as evidenced by antibody inhibition.
  • This work provides a foundation for detailed functional and structural analysis of the RP-A 32-kDa subunit.

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