UC Santa Barbara

Site-specific DNA binding by proteins is critical for diverse biological processes, including gene regulation, DNA repair, DNA replication, immune response, and others. In most cases, this binding is preceded by facilitated diffusion, where the protein passively moves along nonspecific DNA (DNA lacking particular binding sites) in search of its site(s). Details concerning the mechanisms of how proteins move along DNA remain unclear yet actively investigated. Several reports claim that facilitated diffusion relies primarily on sliding and hopping processes, where the protein moves along the trajectory of the DNA helix. However, these mechanisms involve redundant searches over local regions (up to hundreds of base pairs), while site finding in vivo requires searches of hundreds of thousands of base pairs. We "rethink" facilitated diffusion in two ways: 1) what are the mechanisms available for facilitated diffusion? And 2) how are these mechanisms driven by proteins' specific biological context?

We investigate the facilitated diffusion properties of E. coli DNA adenine methyltransferase (Dam), which methylates palindromic 5'-GATC-3' sites on the N6 position of adenine. We use an in vitro steady-state processivity assay, where the ability of the enzyme to catalyze multiple methylations within a single binding event is quantified. Our results are suggestive of a new mechanism, intersegmental hopping, where proteins hop between regions of a single DNA molecule that are looped together. This mechanism allows for efficient long-range movements, and is similar to other ambiguously described looping mechanisms proposed in recent reports. Dam's low cellular copy number and need to move along the entire bacterial chromosome (i.e. its biological context) is ideally accommodated by this mechanism, as we imagine will be true for other proteins with low copy numbers and rare sites.

We also demonstrate that EcoRI ENase uses an extreme sliding mechanism, previously hypothesized to be unlikely. EcoRI ENase and EcoRV ENase both cut incoming phage DNA as part of type II restriction modification systems, and are shown here to use distinct facilitated diffusion mechanisms. We argue that their site finding mechanisms are complimentary, ensuring robust phage defense, again demonstrating the connection between facilitated diffusion and biological context.

Furthermore, we show that Dam is able to move along DNA in spite of pre-incubated protein roadblocks. Provocatively, DNA-loop inducing roadblocks, such as the histone-like Lrp, can improve the translocation process by specifically enhancing intersegmental hopping. Therefore, this mechanism is proposed as a general way for proteins to move along the crowded, compacted genomic DNA landscape.

Dam was previously demonstrated to undergo intrasite processivity, where the methylation of both strands within a single GATC site proceeds by a single Dam enzyme dramatically reorienting itself while switching between strands. Here, we show that intrasite processivity is only possible on long stretches of DNA when GATC sites are clustered. GATC sites are clustered in most known and putative Dam methylation dependent epigenetic operons, which involve transitions from uniquely unmethylated GATC sites to fully methylated ones. Intrasite processivity is therefore postulated as necessary for epigenetic gene regulation by Dam.