DNA Repair Excision Nuclease Attacks Undamaged DNA

Abstract

Nucleotide excision repair is a general repair system that eliminates many dissimilar lesions from DNA. In an effort to understand substrate determinants of this repair system, we tested DNAs with minor backbone modifications using the ultrasensitive excision assay. We found that a phosphorothioate and a methylphosphonate were excised with low efficiency. Surprisingly, we also found that fragments of 23-28 nucleotides and of 12-13 nucleotides characteristic of human and Escherichia coli excision repair, respectively, were removed from undamaged DNA at a significant rate. Considering the relative abundance of undamaged DNA in comparison to damaged DNA in the course of the life of an organism, we conclude that, in general, excision from and resynthesis of undamaged DNA may exceed the excision and resynthesis caused by DNA damage. As resynthesis is invariably associated with mutations, we propose that gratuitous repair may be an important source of spontaneous mutations.Nucleotide excision repair is a general repair system that removes damaged bases from DNA by dual incisions of the damaged strand at some distance from the lesion, releasing the damaged base in the form of 12-13-mers in prokaryotes and 24 -32-mers in eukaryotes (1, 2). It is the major repair system for bulky base adducts, but it also acts on nonbulky lesions such as oxidized or methylated bases and, as such, functions as a backup system for DNA glycosylases, which have restricted substrate ranges (3, 4).The wide substrate spectrum of the excision nuclease raises two interrelated questions: what is the substrate range of the enzyme system and how does the enzyme recognize substrate? Both of these questions have been addressed in numerous studies, and at present we have a basic understanding of damage recognition in both prokaryotes and eukaryotes (1, 2, 5, 6). With regard to substrate range, its limits remain to be defined. The excision nuclease, which originally was thought to be specific for bulky lesions, was later found to excise nonbulky adducts such as methylated bases but, apparently, failed to excise nucleotides with backbone modifications such as the C4Ј pivaloyl adduct (5, 6). With the availability of more efficient in vitro systems (4, 7, 8) we decided to re-examine the question of recognition of backbone modifications. We found that both phosphorothioate and methylphosphonate backbone modifications were recognized as substrates by the human excision nuclease. This, in turn, led us to take a closer look at the effect of the enzyme system on undamaged DNA. We find that both the human and the Escherichia coli excision nucleases excise oligomers of 23-28 and 12-13 nucleotides, respectively, from undamaged DNA. This gratuitous excision and the inevitable repair synthesis that must follow could be potential sources of spontaneous mutations. Our data suggest that even in nondividing cells in which there is no DNA replication, there can be significant DNA turnover due to gratuitous excision and resynthesis and that this gratuitous "repair" may ca...

Sticky DNA Formation in Vivo Alters the Plasmid Dimer/Monomer Ratio

“…We propose that the recognition of DNA distortions in the sticky DNA leads to the enhancement of the D/M ratio in vivo by accelerating k 3 . These data are consistent with previous observations on the interactions of the NER system with undamaged DNA (63) and with triplexes (64 -66) as well as with the effect of the methyl-directed mismatch repair and NER systems on the behavior of plasmids harboring different types of triplexes (32).…”

A mathematical model for human nucleotide excision repair: Damage recognition by random order assembly and kinetic proofreading

“…As this parameter directly controls the number of lesions proceeding through all three kinetic proofreading steps effectively setting the accuracy of kinetic proofreading, its significant impact on the model is unsurprising. The parameters k 1 (the rate at which the first kinetic proofreading step occurs), Coop (backward cooperativity was used in this study), and Pr H (the probability that a repair factor binding to damaged DNA binds at the damage site) all have moderate sensitivity values (0.99, 0.65, and 0.44 respectively) which is consistent with the fact that while all of these parameters directly impact the rate at which the excision complexes form; k 1 and Pr H only affect the model in one location each and the cooperativity constant, while impacting the model at several different locations, affects only the dissociation rates of proteins. As the dissociation rates are small to begin with, the impact of changing them is likewise small.…”

“…It acts on lesions such as photoproducts that distort the helix drastically, on Pyr<>Pyr that makes more subtle structural changes in DNA, on methylated purines and reduced pyrimidines that cause yet subtler structural changes, and even on undamaged DNA ( [9], [1]). Many of these lesions including the classic substrate for excision repair, the Pyr<>Pyr, cannot be discriminated from undamaged DNA by the three damage recognition factors, RPA, XPA, and XPC [19].…”

Xeroderma Pigmentosum Complementation Group A Protein (XPA) Modulates RPA-DNA Interactions via Enhanced Complex Stability and Inhibition of Strand Separation Activity

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