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Coordinated regulation of replication protein A activities by its subunits p14 and p32

Klaus Weisshart1, Pavel Pestryakov, Richard W P Smith

  • 1Institute of Molecular Biotechnology, Beutenbergstrasse 11, Jena 07745, Germany.

The Journal of Biological Chemistry
|June 19, 2004
PubMed

Insights

Replication protein A (RPA) requires all three subunits for efficient DNA binding and replication. Removing the smallest subunit (p14) impairs DNA polymerase interactions and SV40 DNA replication.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Replication protein A (RPA) is crucial for DNA metabolism and signaling in eukaryotes.
  • The precise role of RPA's smallest subunit, p14, remains largely undetermined.
  • Understanding RPA subunit functions is key to elucidating DNA replication mechanisms.

Purpose of the Study:

  • To investigate the function of the smallest RPA subunit, p14.
  • To characterize a dimeric RPA complex (RPADeltap14) lacking p14.
  • To determine the necessity of all three RPA subunits for DNA replication.

Main Methods:

  • Production and characterization of a p14-deficient RPA complex (RPADeltap14).
  • Analysis of single-stranded DNA binding affinity and mode.
  • Assessment of primer-template junction recognition and DNA polymerase interactions.
  • Evaluation of RPADeltap14's ability to support SV40 DNA replication in vitro.

Main Results:

  • RPADeltap14 exhibited altered single-stranded DNA binding compared to the heterotrimeric complex.
  • The p14 and p32 subunits cooperate to stabilize the p70 subunit.
  • RPADeltap14 could not support cell-free SV40 DNA replication, failing in primer synthesis and elongation.
  • Despite supporting DNA unwinding and interacting with SV40 T antigen and DNA polymerase alpha-primase, RPADeltap14 was insufficient for replication.

Conclusions:

  • All three subunits of RPA are essential for efficient binding and proper positioning on single-stranded DNA.
  • The p14 subunit plays a critical role in supporting DNA replication, particularly primer synthesis and elongation.
  • The study highlights the indispensable nature of the complete heterotrimeric RPA complex for eukaryotic DNA replication.

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