The F plasmid traY gene product binds DNA as a monomer or a dimer: structural and functional implications

The F factor traY gene product (TraYp) is a site-specific DNA-binding protein involved in initiation of DNA transfer during bacterial conjugation. The sequence of TraYp exhibits a unique direct-repeat structure predicted to have a ribbon-helix-helix DNA-binding motif in each repeat unit. The stoichiometry of TraYp binding to DNA was determined to further support the hypothesis that TraYp is a member of the ribbon-helix-helix family of DNA-binding proteins. A gluta-thione-S-transferase-traY fusion protein was purified and shown to possess almost wild-type DNA-binding activity. DNA-binding experiments were performed in which the DNA ligand was incubated with either the fusion protein, the wild-type protein, or both. The results indicate that TraYp can bind DNA as a monomer or a dimer. Thus a TraYp monomer folds into a stable three-dimensional structure similar to that of a dimer of the ribbon-helix-helix proteins Arc or Mnt. A homology model of a TraYp monomer has been constructed using the co-crystal structure of Arc bound to DNA as a template to provide additional support for this conclusion. In addition, we have shown that an origin of the transfer-deletion mutant lacking approximately half of the TraYp-binding site can only be bound by a monomer of TraYp. The functional implications of this result are discussed.     

The herpes simplex virus processivity factor, UL42, binds DNA as a monomer

The processivity subunit of the herpes simplex virus DNA polymerase, UL42, is a monomer in solution. However, UL42 is structurally similar to sliding clamp processivity factors, such as PCNA, which encircle DNA as a multimeric ring. We used chemical crosslinking and electrophoretic mobility-shift assays to investigate whether UL42 oligomerizes upon DNA binding. UL42 did not form intermolecular crosslinks upon treatment with glutaraldehyde in the presence of DNA, whereas proteins that are known to be multimers in solution were successfully crosslinked by this treatment. This result suggests that UL42 does not form multimers on DNA. We next analyzed the composition of UL42:DNA complexes using electrophoretic mobility-shift assays. UL42 was mixed with a maltose-binding protein-UL42 fusion protein before being added to DNA. The patterns of electrophoretic mobility of the resultant protein:DNA complexes were those predicted if each isoform of UL42 binds to DNA as a monomer. From this result and the failure of UL42 to form crosslinks, we infer that UL42 binds DNA as a monomer.     

The mitotic regulator Survivin binds as a monomer to its functional interactor Borealin

Survivin is a member of the IAP (inhibitor of apoptosis) protein family, defined in part by the presence of a zinc-binding baculoviral inhibitory repeat (BIR) domain. Most BIR domains bind short sequences beginning with alanine, and in this manner, they recognize and block the action of key targets in apoptotic pathways. However, Survivin binds only very weakly to typical IAP ligands. Unique features of Survivin are the long C-terminal helix following the BIR domain and a short segment (linking the helix and BIR domains) that mediates Survivin homodimerization. Despite this detailed knowledge of the structure of Survivin itself, there is a current lack of understanding about how Survivin recognizes cellular binding partners, and consequently, many questions about Survivin function remain unanswered. We determined two co-crystal structures of Survivin and a minimal binding fragment from the chromosomal passenger protein Borealin, a well validated functional interactor. The interaction between Survivin and Borealin involves extensive packing between the long C-terminal helix of Survivin and a long Borealin helix. Surprisingly, an additional important interaction occurs between the Survivin homodimerization interface and a short segment of Borealin. This segment both structurally mimics and displaces one Survivin monomer. The relevance of this unexpected interaction was tested by mutagenesis of two key Borealin residues. Mutant Borealin introduced into HeLa cells failed to localize properly during mitosis and also caused mislocalization of other chromosomal passenger proteins. This suggests that the mutant is dominant-negative and confirms the functional importance of the interaction surface identified in the crystal structures.     

Oct-2 DNA binding transcription factor: functional consequences of phosphorylation and glycosylation

Phosphorylation and O-GlcNAc modification often induce conformational changes and allow the protein to specifically interact with other proteins. Interplay of phosphorylation and O-GlcNAc modification at the same conserved site may result in the protein undergoing functional switches. We describe that at conserved Ser/Thr residues of human Oct-2, alternative phosphorylation and O-GlcNAc modification (Yin Yang sites) can be predicted by the YinOYang1.2 method. We propose here that alternative phosphorylation and O-GlcNAc modification at Ser191 in the N-terminal region, Ser271 and 274 in the linker region of two POU sub-domains and Thr301 and Ser323 in the POUh subdomain are involved in the differential binding behavior of Oct-2 to the octamer DNA motif. This implies that phosphorylation or O-GlcNAc modification of the same amino acid may result in a different binding capacity of the modified protein. In the C-terminal domain, Ser371, 389 and 394 are additional Yin Yang sites that could be involved in the modulation of Oct-2 binding properties.     

The ColE2-P9 Rep protein binds to the origin DNA as a monomer

The Rep proteins of some plasmid replicons have two functions. Dimers bind to the operator sequences acting as auto-repressors, whereas monomers bind to the iterons to initiate replication of DNA. The ColE2 Rep proteins are present mostly in a dimeric form with some multimers larger than dimers in solution, while the form of Rep binding to Ori is not known. We used an EMSA-based method to determine the molecular weight of Rep in the Rep-Ori complex. The result suggested that Rep binds to Ori as a monomer. In addition, the result of EMSA using the Rep protein fused with the maltose binding protein and the His6-tag also supported this conclusion. We proposed that dimerization of Rep might probably be involved in keeping the copy number of the ColE2 plasmid at the normal low level by limiting the amount of active monomeric forms of Rep in the host cell.     

Replication regulation of Vibrio cholerae chromosome II involves initiator binding to the origin both as monomer and as dimer

The origin region of Vibrio cholerae chromosome II (chrII) resembles plasmid origins that have repeated initiator-binding sites (iterons). Iterons are essential for initiation as well as preventing over-initiation of plasmid replication. In chrII, iterons are also essential for initiation but over-initiation is prevented by sites called 39-mers. Both iterons and 39-mers are binding sites of the chrII specific initiator, RctB. Here, we have isolated RctB mutants that permit over-initiation in the presence of 39-mers. Characterization of two of the mutants showed that both are defective in 39-mer binding, which helps to explain their over-initiation phenotype. In vitro, RctB bound to 39-mers as monomers, and to iterons as both monomers and dimers. Monomer binding to iterons increased in both the mutants, suggesting that monomers are likely to be the initiators. We suggest that dimers might be competitive inhibitors of monomer binding to iterons and thus help control replication negatively. ChrII replication was found to be dependent on chaperones DnaJ and DnaK in vivo. The chaperones preferentially improved dimer binding in vitro, further suggesting the importance of dimer binding in the control of chrII replication.