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

Abstract

The energetic nature of the interactions of DNA base excision repair glycosylases with undamaged and damaged DNA and the nuclear environment are expected to significantly impact the time it takes for these enzymes to search for damaged DNA bases. In particular, the high concentration of monovalent ions, macromolecule crowding, and densely packed DNA chains in the cell nucleus could alter the search mechanisms of these enzymes as compared to findings in dilute buffers typically used in in vitro experiments. Here we utilize an in vitro system where the concerted effects of monovalent ions, macromolecular crowding and high concentrations of bulk DNA chains on the activity of two paradigm human DNA glycosylases can be determined. We find that the energetic nature of the observed binding free energies of human 8-oxoguanine DNA glycosylase (hOGG1) and human uracil DNA glycosylase (hUNG) for undamaged DNA are derived from different sources. While hOGG1 uses primarily non-electrostatic binding interactions with non-specific DNA, hUNG uses a salt-dependent electrostatic binding mode. Both enzymes turn to a non-electrostatic mode in their specific complexes with damaged bases in DNA, which enhances damage site specificity at physiological ion concentrations. Neither enzyme was capable of efficiently locating and removing their respective damaged bases in the combined presence of physiological ions and a bulk DNA chain density approximating that found in the nucleus. However, the addition of an inert crowding agent to mimic macromolecular crowding in the nucleus largely restored their ability to track DNA chains and locate damaged sites. These findings suggest how the concerted action of monovalent ions and crowding could contribute to efficient DNA damage recognition in cells.

AID and Reactive Oxygen Species Can Induce DNA Breaks within Human Chromosomal Translocation Fragile Zones

“…The U’s generated by AID are removed by uracil DNA glycosylase (Ung1) at a rate that can be orders of magnitude more efficient than removal of, for example, the oxidized lesion 8-hydroxyguanine (8OHG) by 8OHG glycosylase (Ogg1) (Cravens and Stivers, 2016). Also, Ung1 can act on both ssDNA and dsDNA whereas Ogg1 recognizes the 8OHG:C base pair in duplex DNA (Friedman and Stivers, 2010).…”

N-terminal domain of human uracil DNA glycosylase (hUNG2) promotes targeting to uracil sites adjacent to ssDNA–dsDNA junctions

“…In the presence of hUNG2 alone, all of the DNA fragments were approximately equal in intensity, indicating that U21 and U45 were kinetically equivalent under these conditions. The lack of discrimination between uracil sites was anticipated based on previous studies that measured hUNG2 activity on DNA duplexes containing two uracil bases at various spacings ( 16 , 17 , 20 , 26 ).…”

Measurement of nanoscale DNA translocation by uracil DNA glycosylase in human cells

“…Many enzymes that act on DNA substrates without the involvement of an energy cofactor have been characterized as proccesive in vitro , where the enzyme can stochastically translocate along a DNA chain before locating a specific target site by sliding or hopping ( 1 – 8 ). Despite the prevalence of these in vitro observations, it is important to keep in mind that enzymes act in a complex cellular environment that differs substantially from test tube conditions.…”

“…Of note, the cellular environment consists of high inorganic ion and metabolite concentrations ( 9 ), lower dielectric properties ( 10 ), higher bulk viscosity ( 11 , 12 ), and the presence of high concentrations of macromolecules which consume available volume (‘molecular crowding’) ( 13 – 15 ). These variables within the cellular environment can have a substantial effect on the ability of an enzyme to scan a DNA chain in a processive manner ( 1 , 13 ).…”

“…Human uracil DNA glycosylase (hUNG2) is a highly-conserved DNA repair enzyme that excises uracil from U/A and U/G base pairs and initiates the process of uracil base excision repair in all organisms ( 16 – 20 ). Like many human DNA glycosylases ( 1 , 2 , 20 – 22 ), hUNG2 has been found to translocate on DNA in vitro , which facilitates the detection and excision of clustered uracils present in a DNA molecule ( 1 , 23 ). The ability of hUNG2 to translocate on DNA in vitro is substantially impacted by both molecular crowding and high ion concentrations that mimic those found in the cellular environment ( 1 , 24 ).…”