Introduction Direct damage reversal (DDR) mechanisms correct DNA lesions without trimming the DNA backbone. here the O6-alkylguanine DNA alkyltransferase (AGT) is usually a highly-conserved protein responsible for direct repair of alkylated guanine and to a lesser degree thymine bases [3 5 In addition to protecting the genome under normal conditions AGT has also recently become a highly interesting target for inhibitor development because it interferes with the deliberate application of DNA alkylation during chemotherapy. Inhibitors of AGT are currently in clinical trial with the aim of mitigating this result of AGT action [1 11 Human AGT is usually a small monomeric protein (Mr ~ 21.5 kDa) . Crystal structures of AGT are available for the and resistance to alkylating brokers . Cooperative interactions are frequently encountered in protein-DNA complexes [31-40]. The mechanisms of these cooperative interactions are as diverse as their target functions which include modulation of DNA-affinity [34-36] stabilization of DNA superstructures [37 38 and regulation of enzyme activity . SB 334867 Atomic pressure microscopy (AFM) imaging has been applied to study a number of cooperative protein-DNA systems ranging from dimeric protein complexes to long multimeric nucleofilaments [31 38 AFM provides direct access to structural features of cooperative protein-DNA complexes at the level of the individual molecules and can provide data essential for an understanding of the underlying mechanisms. Importantly the concentration-dependent length of the protein complexes around the DNA which is usually directly related to the intrinsic DNA affinity and the cooperativity of the interactions can be derived from AFM images. These characteristics will be discussed in the context of the AGT-DNA system below. Models of cooperative AGT assemblies have been built based on the crystal structures of DNA-bound AGT monomers [2 21 and taking into account observed binding densities and comparable affinities for single-stranded and duplex substrates [26 27 In these models (for example Physique 2) each protein monomer in the cooperative SB 334867 unit is usually rotated with respect to its neighbors by 138° (corresponding to an apparent binding site size of 4 bp). A surface-charge representation of the cooperative complex model discloses a positively-charged channel of AGT monomers that is occupied by the negatively charged DNA. In this model each nth and (n+3)rd monomer in the complex interact directly but contacts between adjacent monomers are poor or non-existent. This model of non-specific cooperative AGT-DNA lesion search complexes can be tested by single molecule AFM imaging. Physique 2 Model of a cooperative AGT-DNA complex 2 Rabbit polyclonal to TRIM3. Model description of non-specific cooperative protein-DNA interactions The McGhee-von Hippel binding model is usually often used to characterize non-specific cooperative protein-DNA interactions [41 42 While this model simplifies some of the complex biological processes it provides useful guidelines for an overall description and comparison of cooperatively interacting systems. Simplifying assumptions used in SB 334867 the model include a single binding mode for all those protein molecules within the cooperative unit characterized by a unique binding site size can be obtained experimentally by sedimentation equilibrium centrifugation or gel mobility shift techniques and evaluated using a variant of the Scatchard equation (Equation 1) [26 27 is the protein binding density around the DNA and [P] is the free protein concentration supporting the equilibrium. When binding is usually defined according to the McGhee-von Hippel model the imply cooperative cluster size is usually predicted to be : = (6.81 ± 0.14) and (6.32 ± 0.12) bp = (7960 ± 916) and (9667 ± 1499) and = (44.2 ± 3.8) and (35.9 ± 6.8) were obtained respectively from sedimentation equilibrium analytical ultracentrifugation data . The observed limiting binding site sizes were larger than the value of ~4 bp/protein obtained with shorter DNA oligonucleotides . SB 334867 This likely reflects packing inhomogeneities that occur when the substrate is usually large and which are largely absent near binding saturation when short substrates are used. The dependence of cluster size on AGT concentration predicted by McGhee-von Hippel theory using these.