DNA Adenine Methyltransferase Facilitated Diffusion Is Enhanced by Protein–DNA “Roadblock” Complexes That Induce DNA Looping
journal contributionposted on 07.04.2015, 00:00 by Adam J. Pollak, Norbert O. Reich
The genomes of all cells are intimately associated with proteins, which are important for compaction, scaffolding, and gene regulation. Here we show that pre-existing protein–DNA complexes (roadblocks) diminish and―interestingly―enhance the ability of particular sequence-specific proteins to move along DNA to locate their binding sites. We challenge the bacterial DNA adenine methyltransferase (Dam, recognizes 5′-GATC-3′) with tightly bound EcoRV ENase–DNA complexes, which bend DNA. A single EcoRV roadblock does not alter processive (multiple modifications) methylation by Dam. This result disfavors a reliance on heavily touted mechanisms involving sliding or short hops for Dam. Specific conformations of two EcoRV roadblocks cause an increase in processivity. The histone-like leucine-responsive regulatory protein (Lrp) binds DNA nonspecifically as an octamer, and also increases Dam’s processivity. These results can be explained by our prior demonstration that Dam moves over large regions (>300 bp) within a single DNA molecule using an “intersegmental hopping” mechanism. This mechanism involves the protein hopping between looped DNA segments. Both roadblock systems can cause the DNA to loop and therefore facilitate intersegmental hopping. For Lrp, this only occurs when the Dam sites are separated (by >134bp) such that they can be looped around the protein. Intersegmental hopping may well be a general mechanism for proteins that navigate long distances along compacted DNA. Unlike Dam, EcoRI ENase (recognizes 5′-GAATTC-3′) relies extensively on a sliding mechanism, and as expected, Lrp decreases its processivity. Our systematic use of protein roadblocks provides a powerful strategy to differentiate between site location mechanisms.
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Lrp decreasesDNA segmentsbinding sitesGATCEcoRI ENaseEcoRV roadblockresult disfavorsprotein roadblocksDNA Adenine Methyltransferase Facilitated Diffusionroadblock systemssite location mechanismsgene regulationEcoRV roadblocks causeDNA adenine methyltransferaseInduce DNA LoopingThe genomesSpecific conformationsDNA moleculeDam sitesDam movesGAATTC