The Action Pattern of Mammalian Deoxyribonuclease IV

Abstract

The mode of action of an exonuclease that probably is involved in DNA repair, mammalian DNase IV, has been studied. By kinetic measurements with both uniformly and terminally labeled polynucleotides it is shown that the enzyme breaks several adjacent phosphodiester bonds each time it binds to its substrate, but that the number of hydrolytic events is small compared to the degree of polymerization of the substrate. At 37" the enzyme releases 50-200 nucleotides in an exonucleolytic fashion a t each encounter with native DNA. The significance of these observations in relation to DNA repair in vivo is discussed.Enzymes that degrade linear polymers often act in a step-wise fashion by releasing one residue a t a time from one of the chain ends. An enzyme of this type is usually assumed to attack polymer chains in a random fashion, and to dissociate from its substrate after a single hydrolytic event. I n apparent contrast to such a mechanism of action, however, a number of exonucleases have recently been found to remain bound to their substrate during hydrolysis, so that one polynucleotide chain is completely degraded before the next one is attacked [l-41. An action pattern of this type has been referred to as single chain action I n the conventional Michaelis theory, an enzyme (E) and a substrate (S) are postulated to react in a reversible way to form a complex (ES), which then reacts to give products (P) and free enzymeIn the case of a polymer and an enzyme that remain associated and just move relative to each other during hydrolysis [5--81, the system is better represented by a modified expression I n this expression [5,8], the kinetic constants k,, Ll, and k, govern the rate of formation of an ES complex, its lifetime, and the rate of product formation, respectively. It further follows that the average number of hydrolytic events a t each encounter beUnusual Abbreviation. Poly[d(A-T)], an alternating double-stranded copolymer of deoxyadenylate and deoxythymidylate.All tritium-labeled polymers are labeled in the thymine residues.tween the enzyme and its polymer substrate is obtained directly from the ratio k, : k-l. Thus the two modes of action referred to above, random enzymatic attack at all available chain ends us. degradation of one complete polymer chain at a time, are not necessarily mutually exclusive ; they can instead be regarded as two extreme cases of the same type of reaction mechanism [5,8]. In the former case, the rate constant k-, should be larger than k,, while the reverse should be true in the latter case. It might also be expected that polymerdegrading enzymes with a reaction mechanism intermediary between these two extremes would exist. I n a detailed study of the kinetic properties of 8-amylase) a plant enzyme that releases maltose residues from the non-reducing end of an amylose chain, such an intermediary action pattern was indeed observed in that a few maltose residues were removed at each effective enzyme-substrate encounter [5,9].In the present work, the mode of degradation of DNA and polyde...

Involvement of single-stranded tails in homologous recombination of DNA injected into Xenopus laevis oocyte nuclei.

“…ExollI-treated pRW4 was incubated with Klenow polymerase in the presence of all four dideoxynucleotides for 10 min at room temperature. The 50-,u reaction mixture contained 50 mM Tris (pH 7.5), 10 mM MgCl2, 1 mM dithiothreitol, 25 [29]. The demonstration later in this report that 5' tails are rapidly made flush in oocytes provides additional justification for calling XhoI ends flush in this context.)…”

“…If, as our data suggest, the 5'--3' exonuclease plays a central role in this type of recombination, it may be a ubiquitous component of eukaryotic DNA metabolism that has generally escaped attention. Interestingly, a similar activity purified from rabbit liver was described about 20 years ago (26) and was postulated to be involved in the repair of UV-induced damage (25).…”

Enzymatic Repair of DNA

“…The catalytic properties of two generally distinguishable but functionally related exonucleases capable of excising pyrimidine dimers have been identified in M. luteus (72), E. coli (154), T4-infected E. coli (127,155), rabbit liver nuclei (156,157), human placenta (158), and mammalian cell lines (118). Both types of exonucleases, exonuclease and exonuclease V|I, have specificities limited to denatured DNA and are capable of hydrolyzing UV damaged and undamaged substrates at comparable rates and to the same extent (159).…”

A Yeast Replicative Helicase, Dna2 Helicase, Interacts with Yeast FEN-1 Nuclease in Carrying Out Its Essential Function

“…It was therefore relevant that a 5Ј33Ј exoendonuclease activity is present in Dna2 immunoprecipitates and in immunoaffinity-purified Dna2 protein samples (2,3). Mammalian DNase IV (FEN-1 or MF1) (7,10,14,22,23) and the S. cerevisiae RAD27/RTH1 product (ORF YKL510) (9,11,19,24) are two previously described exo-endonucleases with substrate specificities similar to that of the Dna2-associated nuclease activity. (Despite the fact that DNase IV was the first name given to this nuclease, to avoid confusion and reflect a number of recent publications, we refer to this enzyme as yeast FEN-1 and to the yeast gene as RAD27/RTH1 in this paper.)…”