Significant effort has been made to develop synthetic metal complexes that hydrolyze DNA. Here we report a new dicerium complex, Ce(2)(HXTA) (HXTA = 5-methyl-2-hydroxy-1,3-xylene-alpha,alpha-diamine-N,N,N',N'-tetraacetic acid), which can hydrolyze DNA at pH 8 and 37 degrees C. This complex hydrolyzes DNA restriction fragments to give products with high regioselectivity, affording >90% 5'-OPO(3) and 3'-OH ends, like the products of DNA hydrolyzing enzymes. Ce(2)(HXTA) also hydrolyzes Litmus 29 plasmid DNA to afford both nicked and linear DNA. Analysis of the relative amounts of supercoiled, nicked, and linear DNA present show that there is one double-strand cleavage per ten single-strand cleavages, indicating that the linear DNA formed cannot be the result of two random single-strand cleavage events. The kinetics of nicked and linear DNA formation are comparable, both being associated with apparent first-order rate constants of approximately 1 x 10(-)(4) s(-)(1) for complex concentrations of 10(-)(5)-10(-)(4) M. These observations suggest that similar factors affect the hydrolysis of the first and second DNA strands and that cleaving the phosphodiester bond is likely the rate determining step in both cases. This is the first detailed study of a metal complex shown to mimic DNA hydrolases in their capability to effect double-strand DNA hydrolysis regioselectively at the 3'-O-P bond.

Efficient DNA cleavage with an iron complex without added reductant Roelfes, Gerard; Branum, M.E.; Wang, L.; Que, L.; Feringa, B.L.; Que, Jr.

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...

Although there has been progress in developing artificial hydrolytic DNA cleaving agents, none of these has been shown to carry out the double-strand hydrolysis of DNA. We demonstrate that La(III) or Ce(IV) combined with the ligand 1,3-diamino-2-hydroxypropane-N,N,N', N'-tetraacetate (HPTA) in a 2 : 1 ratio can efficiently cleave supercoiled plasmid DNA at 55 degrees C within a 3-h period. Analysis of end-labeled restriction fragments cleaved by these complexes reveals 3'- and 5'-ends consistent with a hydrolytic mechanism. Unlike for other polydentate carboxylate complexes, plasmid DNA cleavage by La(2)(HPTA) or Ce(2)(HPTA) affords a significant amount of linear DNA with a considerable fraction of the supercoiled form still remaining. This result implies that La(2)(HPTA) and Ce(2)(HPTA) can carry out double-strand cleavage of plasmid DNA. La(2)(HPTA) and Ce(2)(HPTA) represent the first metal complexes demonstrated to be capable of double-strand hydrolytic cleavage of plasmid DNA.

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