Assays for structure-selective DNA endonucleases

Abstract

Structure-selective nucleases perform DNA strand incisions crucial to the repair/resolution of branched DNA molecules arising during DNA replication, recombination, and repair. From a combination of genetics and in vitro nuclease assay studies, we are just beginning to understand how these enzymes recognize their substrates and to identify their in vivo DNA structure targets. By performing nuclease assays on a variety of substrates meant to mimic cellular intermediates, structural features of branched DNA molecules that are important for robust catalysis can be defined. However, since these enzymes often are capable of cleaving a range of DNA structures, caution must be taken not to overemphasize the significance of incision of a certain structure before a careful and detailed kinetic analysis of a variety of DNA substrates with different polarities and structural features has been completed. Here, we provide protocols for the production of a variety of oligo-based DNA joint molecules and their use in endonuclease assays, which can be used to derive the kinetic parameters KM and kcat. Determination of these values for a variety of substrates provides meaningful comparisons that allow inferences to be made regarding in vivo DNA structure target(s).

Figure 1. Comparison of nuclease activity on DNA joint molecules and kinetic analysis

A, Saccharomyces cerevisiae Mus81-Mms4 incises model DNA joint molecules such as a 3′-flapped DNA. Incision is shown to depend on the nuclease activity of the endonuclease, as a purified mutant complex cannot cut DNA (Mus81-dd is Mus81-D414A, Mms4-D415A). B, Fixed time point Mus81-Mms4 nuclease assays for several DNA joint molecules are shown at fixed substrate concentration (50 nM) and a titration of Mus81-Mms4 from limiting concentration (5 nM) to excess concentration (100 nM). C, Incision proficiency on different DNA joint molecules can be quantitated and graphically represented, as in this quantitation of the data in B. D, To perform a reaction time course, aliquots from an ongoing nuclease reaction are removed at determined intervals and stopped. Here, a 3′-FL time course is shown. ‘dn’ represents heat-denatured substrate, demonstrating that the incision product is specific to the enzyme and not time-dependent denaturation of the substrate. When percent substrate cleaved versus time is plotted (reaction progress curves), the initial rate of the reaction can be extrapolated from early points in the time course over the interval when the reaction rate is linear. E, With initial rates determined over a range of substrate concentrations, a plot of initial velocity versus substrate concentration can be used to determine the Michaelis concentration (K_M) and catalytic turnover (k_cat) of the nuclease on substrates it incises.

Figure 2. Incision site mapping by direct comparison to an oligonucleotide size ladder

A, A number of DNA joint molecules are represented on a denaturing PAGE gel, with oligonucleotide size markers (‘L’) representing a series of possible incision site products on the incised strand. B, The oligonucleotides pooled as incision site markers flank the structure’s branch point on the incised strand. C, The fraction of molecules incised at a particular site can be graphed by quantitation of data in A. For the 3′-FL shown, the majority of incision events occurred four nucleotides 5′ of the structure’s branch point.