Efficient second strand cleavage during Holliday junction resolution by RuvC requires both increased junction flexibility and an exposed 5' phosphate

Abstract

Background: Holliday junction (HJ) resolution is a critical step during homologous recombination. In Escherichia coli this job is performed by a member of the RNase H/Integrase superfamily called RuvC, whereas in Schizosaccharomyces pombe it has been attributed to the XPF family member Mus81-Eme1. HJ resolution is achieved through the sequential cleavage of two strands of like polarity at or close to the junction crossover point. RuvC functions as a dimer, whereas Mus81-Eme1 is thought to function as a dimer of heterodimers. However, in both cases the multimer contains two catalytic sites, which act independently and sequentially during the resolution reaction. To ensure that both strands are cleaved before the nuclease dissociates from the junction, the rate of second strand cleavage is greatly enhanced compared to that of the first. The enhancement of second strand cleavage has been attributed to the increased flexibility of the nicked HJ, which would facilitate rapid engagement of the second active site and scissile bond. Here we have investigated whether other properties of the nicked HJ are important for enhancing second strand cleavage.

Principal findings: A comparison of the efficiency of cleavage of nicked HJs with and without a 5' phosphate at the nick site shows that a 5' phosphate is required for most of the enhancement of second strand cleavage by RuvC. In contrast Mus81-Eme1 cleaves nicked HJs with and without a 5' phosphate with equal efficiency, albeit there are differences in cleavage site selection.

Conclusions: Our data show that efficient HJ resolution by RuvC depends on the 5' phosphate revealed by incision of the first strand. This is a hitherto unappreciated factor in promoting accelerated second strand cleavage. However, a 5' phosphate is not a universal requirement since efficient cleavage by Mus81-Eme1 appears to depend solely on the increased junction flexibility that is developed by the first incision.

Figure 1. Cleavage of X0 and X0n (+/− 5′ phosphate at nick site) by RuvC.

(A) Schematic showing the linear duplex products that are generated by the cleavage of X0 or X0n by RuvC. The asterisk indicates the 5′ ³²P label. (B) Native polyacrylamide gel showing the cleavage of X0 and X0n (+/− 5′ phosphate at nick site) by RuvC. Reactions (40 µl) contained 1.3 nM junction DNA and 50 nM RuvC as indicated. Reactions were incubated at 30°C for 30 min before being stopped. (C) Denaturing gel of the same reactions as in A. (D) Schematic showing the core nucleotide sequences in X0 and X0n and the sites of cleavage by RuvC. (E) A comparison of the rates of cleavage of X0 and X0n (+/− 5′ phosphate at nick site) by RuvC. Reactions (70 µl) contained 1.4 nM junction DNA and 10 nM RuvC. Data are the mean of three experiments. (F) A comparison of RuvC's binding affinity for X0 and X0n (+/− 5′ phosphate at nick site) by RuvC. Reaction conditions are described in Materials and Methods.

Figure 2. Cleavage of M1 and M1n (+/− 5′ phosphate at nick site) by RuvC.

(A) Schematic showing the core nucleotide sequences in M1 and M1n. The asterisk indicates the 5′ ³²P label. (B) A comparison of RuvC's binding affinity for M1 and M1n (+/− 5′ phosphate at nick site) by RuvC. Reaction conditions are described in Materials and Methods. (C) Single turnover kinetic analysis of M1 and M1n (+/− 5′ phosphate at the nick site) cleavage by RuvC. The reaction conditions are described in Materials and Methods. The data are the means of three independent experiments, and the error bars represent the standard deviations.

Figure 3. A comparison of the cleavage of X0n (no 5′ phosphate at nick site) and X0n (+ 5′ phosphate at nick site) by S. pombe Mus81-Eme1.

(A) Reactions (20 µl) contained 1.1 nM junction DNA and the indicated amounts of protein, and were incubated at 30°C for 30 minutes before being stopped and run on a 10% native polyacrylamide gel. The schematic on the right-hand side of the panel shows the duplex products that are generated by the cleavage of X0n by Mus81. The asterisk indicates the 5′ ³²P label. (B) Time courses of X0n (+/− 5′ phosphate at the nick site) cleavage by Mus81. Reactions (40 µl) contained 2 nM junction DNA and 0.2 nM Mus81-Eme1. Values are means±standard error of the mean from three independent experiments.

Figure 4. Effect of a 5′ phosphate at the nick site in X0n on cleavage site preference by Mus81-Eme1.

(A) Denaturing gels showing time courses of cleavage at sites a–d in X0n (+/− 5′ phosphate at the nick site) by Mus81. Reactions (40 µl) contained 2 nM junction DNA and 0.2 nM Mus81-Eme1. (B) Schematic showing the core nucleotide sequences in X0n and the main sites of cleavage by Mus81. (C and D) Mean data from three experiments like shown in A. Error bars are omitted for the sake of clarity.