Microscopic mechanism of DNA damage searching by hOGG1

Abstract

The DNA backbone is often considered a track that allows long-range sliding of DNA repair enzymes in their search for rare damage sites in DNA. A proposed exemplar of DNA sliding is human 8-oxoguanine (oG) DNA glycosylase 1 (hOGG1), which repairs mutagenic oG lesions in DNA. Here we use our high-resolution molecular clock method to show that macroscopic 1D DNA sliding of hOGG1 occurs by microscopic 2D and 3D steps that masquerade as sliding in resolution-limited single-molecule images. Strand sliding was limited to distances shorter than seven phosphate linkages because attaching a covalent chemical road block to a single DNA phosphate located between two closely spaced damage sites had little effect on transfers. The microscopic parameters describing the DNA search of hOGG1 were derived from numerical simulations constrained by the experimental data. These findings support a general mechanism where DNA glycosylases use highly dynamic multidimensional diffusion paths to scan DNA.

Comparative Effects of Ions, Molecular Crowding, and Bulk DNA on the Damage Search Mechanisms of hOGG1 and hUNG

“…hOGG1 DNA translocation experiments were carried out with 90mer substrates containing two 8-oxoG residues positioned 10bp and 20bp apart on the same DNA strand (S10 oxoG and S20 oxoG ) 35 . As previously described, translocation between two damage sites within the context of a single DNA chain can occur by either an associative or dissociative pathway 20, 21, 22, 34, 35 .…”

“…Interestingly, the same increase was not observed for a 10 bp spacing ( P trans buffer = 0.46 ± 0.01; P trans PEG = 0.45 ± 0.01) (Figure 3C, red bars). The basis for this result is not known but may arise from the possibility that translocation of hOGG1 over such short distances is inhibited by the time it takes for the bent DNA to relax its normal structure, which may force the enzyme to dissociate and rebind before reaching the second site 35 . The further addition of 0.1 and 1 mM salDNA to the crowded solution containing S20 oxoG reduced P trans by about 50 and 100%, respectively.…”

AP-Endonuclease 1 Accelerates Turnover of Human 8-Oxoguanine DNA Glycosylase by Preventing Retrograde Binding to the Abasic-Site Product

“…This apparent paradox for the passive mechanism can be resolved if the polymeric nature of DNA is considered along with the microscopic dynamic translocation behavior of these enzymes when interacting with specific and nonspecific DNA chains 34,35 In this regard, the macroscopic off rate from a site is determined by the rate of irreversible departure of the enzyme from the site, defined as its equilibration with all other substrate or product molecules in bulk solution. For an enzyme that interacts with DNA, the macroscopic rate also contains the rapid microscopic excursions that an enzyme makes away from a specific site before it irreversibly departs.…”

“…This in essence sets up a rapid but unfavorable equilibrium that may be heavily biased towards the enzyme rebinding the site rather than departing to bulk solution (“retrograde” AP-site binding; Fig. 7) 34 . These dynamic excursions of hOGG1 away from the AP-site provide a transient opportunity for APE1 or TDG to invade the site.…”

A walk along DNA using bipedal migration of a dynamic and covalent crosslinker

“…For these, Brownian diffusion may remain optimum and highly satisfactory as evident from the success of DNA repair. Detection of nucleobase damage is a formidable task yet solved by nature using diffusional rather than directional scanning [27][28][29] . Whether directional or diffusional, transport based on covalent exchange still lacks a readily accessible and easily manipulated molecular surface to guide reaction.…”