The single-stranded DNA-binding protein of Escherichia coli.

The DNA polymerase accessory factor proliferating cell nuclear antigen (PCNA) has been caught in interaction with an ever increasing number of proteins. To characterize the sites and functions of some of these interactions, we constructed four mutants of human PCNA and analysed them in a variety of assays. By targeting loops on the surface of the PCNA trimer and changing three or four residues at a time to alanine, we found that a region including part of the domain‐connecting loop of PCNA and loops on one face of the trimer, close to the C‐termini, is involved in binding to all of the following proteins: DNA polymerase δ, replication factor C, the flap endonuclease Fen1, the cyclin dependent kinase inhibitor p21 and DNA ligase I. An inhibition of DNA ligation caused by the interaction of PCNA with DNA ligase I was found, and we show that DNA ligase I and Fen1 can inhibit DNA synthesis by DNA polymerase δ/PCNA. We demonstrate that PCNA must be located below a 5′ flap on a forked template to stimulate Fen1 activity, and considering the interacting region on PCNA for Fen1, this suggests an orientation for PCNA during DNA replication with the C‐termini facing forwards, in the direction of DNA synthesis.

/pdf/regulation-of-dna-replication-and-repair-proteins-through-28y8l2gl7k.pdf

Regulation of DNA replication and repair proteins through interaction with the front side of proliferating cell nuclear antigen

SSB Functions as a Sliding Platform that Migrates on DNA via Reptation

https://biochem.wisc.edu/sites/default/files/labs/cox/pdfs/ShanJMB1997.pdf

Reca protein filaments : end-dependent dissociation from ssdna and stabilization by reco and recr proteins

Salt-dependent changes in the DNA binding co-operativity of Escherichia coli single strand binding protein

We report a rapid procedure for the large-scale purification of the Escherichia coli encoded single-strand binding (SSB) protein, helix-destabilizing protein which is essential for replication, recombination, and repair processes in E. coli. To facilitate the isolation of large quantities of the ssb gene product, we have subcloned the ssb gene into a temperature-inducible expression vector, pPLc28 [Remaut, E., Stanssens, P., & Fiers, W. (1981) Gene 15, 81-93], carrying the bacteriophage lambda PL promoter. A large overproduction of the ssb gene product results upon shifting the temperature of E. coli strains which carry the plasmid and also produce the thermolabile lambda cI857 repressor. After 5 h of induction, the ssb gene product represents approximately 10% of the total cell protein. The overexpression of the ssb gene and the purification protocol reported here enable one to isolate SSB protein (greater than 99% pure) with final yields of approximately 3 mg of SSB protein/g of cell paste. In fact, very pure (greater than 99%) SSB protein can be obtained after approximately 8 h, starting from frozen cells in the absence of any columns, although inclusion of a single-stranded DNA-cellulose column is generally recommended to ensure that the purified SSB protein possesses DNA binding activity. The ability to easily purify 1 g of SSB protein from 300-350 g of induced cells will facilitate physical studies requiring large quantities of this important protein.

Large-scale overproduction and rapid purification of the Escherichia coli ssb gene product. Expression of the ssb gene under .lambda. PL control

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Large-scale overproduction and rapid purification of the Escherichia coli ssb gene product. Expression of the ssb gene under .lambda. PL control