| Single strand DNA binding proteins, or SSB's, play a crucial part in DNA replication, recombination, and repair for many organisms. The gene 32 protein of bacteriophage T4 (gp32) is well known for its model behavior as a classical SSB. Limited proteolysis of gp32 affords three consecutive domains that are functionally unique---a positively-charged N-domain, a core domain where single-stranded DNA (ssDNA) is bound, and a negatively-charged C-domain (CTD). The C-terminal domain influences heterotypic protein interaction and inhibits gp32's ability to destabilize double-stranded DNA (dsDNA). The C-domain is proposed to weakly bind to the core domain and compete with free ssDNA for binding. In occupying the ssDNA binding region of the core domain, CTD is thought to act as a DNA mimic. This behavior comes from core domain-DNA binding being largely charge dependent (electrostatic interactions). The goal of this investigation is to determine whether the C-domain serves as an internal ssDNA mimic and electrostatic shield by interacting with the core-domain binding cleft.;We have overexpressed and purified slightly lengthened C-domain, and employed a modified zero-length cross-linking protocol to selectively attach CTD to core domain. The reaction resulted in the appearance of core + CTD bands on SDS-polyacrylamide gels. Addition of ssDNA or high salt levels during cross-linking created a competitive binding assay, both of which inhibited cross-linked complex formation. We also demonstrated that cross-linked products reflect the native binding position for the C-domain, which supports our current model, where binding of ssDNA and C-domain to the core-domain is mutually exclusive. |
