Replication protein A is the major single-stranded DNA binding protein detected in mammalian cell extracts by gel retardation assays and UV cross- linking of long and short single-stranded DNA molecules

E. Seroussi, S. Lavi

Research output: Contribution to journalArticlepeer-review

Abstract

Band shift and UV cross-linking assays were used to analyze the major single-stranded DNA (ssDNA) binding activity in lysates of primate and rodent cells. The ssDNA binding activity behaved chromatographically similar to that of replication protein A (RP-A), a multisubunit protein containing three polypeptides of molecular mass 70, 34, and 14 kDa. A 70-kDa protein was found to harbor the ssDNA binding activity when UV cross-linked to long ssDNA or to oligonucleotide probes. Monoclonal antibodies against the 70- and the 34-kDa subunits produced super-gel-shift patterns, demonstrating that the reactive protein is indeed RP-A and that the retarded native binding complex included both subunits. RP-A displayed oligonucleotide-specific binding dependent on oligomer length. Increasing oligonucleotide length led to the formation of slow migrating complexes harboring multiple RP-A molecules, suggesting that an interval of about 20-30 bases is required for the binding of RP-A molecules. While similar binding activity was detected in cell extracts derived from proliferating and quiescent cells, a sharp decline in ssDNA binding activity was observed in the SV40-transformed Chinese hamster cell line 631 following UV irradiation. The nature of this decrease in activity and its possible effect on DNA replication is discussed.

Original languageEnglish
Pages (from-to)7147-7154
Number of pages8
JournalJournal of Biological Chemistry
Volume268
Issue number10
StatePublished - 1993

Fingerprint

Dive into the research topics of 'Replication protein A is the major single-stranded DNA binding protein detected in mammalian cell extracts by gel retardation assays and UV cross- linking of long and short single-stranded DNA molecules'. Together they form a unique fingerprint.

Cite this