Conformational Dynamics of the E. Coli SOS Mutagenesis Protein UmuD

Abstract

The SOS response in Escherichia coli involves the induction of at least 57 genes in response to DNA damage, including the umuD gene. UmuD2 is a homodimer of 139-amino acid subunits that interacts with RecA/ssDNA nucleoprotein filaments, resulting in cleavage of its N-terminal 24-amino acids to yield UmuD’2. The full-length form UmuD plays a role in a primitive DNA damage checkpoint and prevents mutagenesis, whereas the cleaved form facilitates mutagenesis by Y family DNA polymerase UmuC. The goal of our research is to determine the conformation and dynamics of UmuD in order to understand its regulatory role in response to DNA damage. UmuD2 and UmuD’2 are both exceptionally tight dimers, with picoMolar dissociation constants. However, we find that a monomeric variant of UmuD maintains essentially all biological roles of UmuD2. This surprising finding suggests that UmuD can cleave in either a cis or trans conformation. By forming alternate dimeric forms of UmuD, we determined that cleavage in trans is more efficient than cleavage in cis. UmuD2 and UmuD’2 readily exchange monomers to form the heterodimer UmuDD’, which is the most stable dimeric form. We are investigating the kinetics and mechanism of exchange of UmuD dimers and the UmuDD’ heterodimer by using FRET assays and native PAGE. The different dimeric forms of the umuD gene products exchange on the minute time scale, and each dimer can continue to exchange once formed. Designed site-directed mutations are expected to allow us to determine the mechanism of UmuD dimer exchange.