We show here that yDna2 nuclease/helicase contains a four cysteine Fe–S cluster. As the first cysteine residue and the remaining three cysteines are separated by more than 250 amino acids spanning the nuclease active site, the Fe–S cluster may ‘staple’ the nuclease domain to give a unique tertiary structure to Dna2, by analogy to AddB (10
). Interestingly, although the metal domain is found in the N-terminal nuclease domain, mutation of any of the four cysteines that bind iron not only led to reduction of nuclease activity but also reduced the ATPase of the C-terminal helicase domain. Although the mutations diminished both the nuclease and helicase activities, surprisingly, the DNA binding affinity of the mutants was similar to WT. Additionally, the conformation of the apoprotein was unchanged as determined by protease sensitivity, expression and recovery of purified protein and ability to act as a negative factor in vivo
. In view of these observations, we propose that the defect in the Fe–S cluster disrupts obligatory dynamic conformational alterations during coupled cleavage/translocation reactions. An example of coupling is that binding contacts made by the nuclease could increase the translocation processivity of the helicase. The likely interaction and interdependence of nuclease and helicase functions provide an explanation for why we were not able to detect nuclease activity in a fragment of Dna2 containing amino acids 1–963 nor ATPase in a partial protein from amino acids 964–1522 in the past, i.e. because their activities stimulate each other (15
). For instance, tightly bound nuclease might increase the processivity of the helicase. Others have also tried and failed to express nuclease and ATPase activities independently (26
). Biochemical evidence for coupling has been presented previously by showing that helicase stimulates nuclease activity on flap substrates containing secondary structure (18
), and the experiments suggested strongly that the active helicase and nuclease have to be in the same polypeptide, supporting conformational coupling (27
). However, the structural basis for these observations remains elusive.
Our results define a novel paradigm of Fe–S domain function that not only resembles but also differs from other proteins containing fused nuclease and helicase/translocase domains. Based on the AddB protein (10
), we had expected that the C to A mutations would seriously impact DNA binding. This was not the case, possibly because the Fe–S cluster of AddB is important for binding dsDNA ends, and Dna2 only binds flaps or unwound ends. The defect in binding dsDNA in AddB mutants thus indirectly inactivates the dsDNA-dependent AddAB ATPase/translocase, associated with AddA. However, the Fe–S mutations do not alter the ssDNA-dependent ATPase of AddAB (10
). Characteristics of Dna2 more closely resemble those of the HsdR subunit of the type 1 restriction enzymes, which contain N-terminal nuclease and C-terminal translocase domains. In the HsdR subunit of EcoR124I, mutations in the catalytic motifs II and III of the nuclease have dramatic effects on the C-terminal translocase activity. The similarity lies in the implied long-range interaction between the nuclease and translocase activities. Remarkably, this cross-talk is not mediated by and Fe–S cluster in HsdR. A WT Dna2 protein lacking Fe may mimic EcoR124I, in that there may be some residual interaction. The Dna2 Fe–S cluster also differs from that in the xeroderma pigmentosum group D (XPD) and FancJ families, where the cluster contains four closely associated cysteines inserted in helicase domain 1, contributing to an arch that is important for coupling ATPase and unwinding (5
). Yet, another 4C motif with four closely linked cysteines is located in DNA polymerases; and, in DNA polymerase delta, the cluster stabilizes subunit interactions and replisome stability but does not have a long-range effect on polymerase catalysis (29
The helicase domain also reciprocally regulates the nuclease domain. A mutation in helicase motif 1, K1080E, which affects but does not abolish the ATP β- and γ-phosphate binding, did not inactivate but significantly changed the nuclease. Normally, the nuclease is inhibited by ATP. However, in the K1080E protein, the nuclease is highly stimulated by ATP (15
). This resembles the ATP-dependent stimulation of Dna2 exonuclease in the presence of Mn+2
instead of Mg+2
). ATP also inhibited strand annealing in Dna2-K1080E (30
). Remarkably, the N-terminal nuclease domain of a bacterial DinG protein has also recently been shown to be regulated by a C-terminal ATPase (31
). Finally, in vivo
, a nuclease active site mutant, is toxic, but dna2-D675A,1080E
is viable and X-ray resistant, strongly supporting a model in which helicase must be coordinated with nuclease for viability. Perhaps, helicase makes a product that requires nuclease for resolution (32
The temperature-sensitive phenotype of the dna2-1
mutation carrying the P504S substitution had always been puzzling, in that the location of the mutation, roughly 250
bp from the nuclease active site and more than 1
kb away from the helicase domain, had a dramatic effect on both nuclease and helicase activities. Other mutations introduced in the nuclease or helicase motifs affected only one, but not the other enzymatic activities. Based on the following current observations, we now propose that the temperature-sensitive phenotype of the dna2-1
mutation is based on destabilization of the Fe–S cluster domain. First, sequence analysis identifies the P504, proximal to the first C of the Fe–S motif, as a conserved residue (). Second, mutational disruption of the Fe–S cluster results in major inhibition of both nuclease and helicase activities of Dna2, similar to the dna2-1
mutation. Third, another dna2
mutation that changes R521 (33
), which is conserved in both Dna2 and AddB, falls in the highly conserved region of the Fe–S motif () and also results in a temperature-sensitive phenotype. We caution, however, that the R521K substitution also carries a G446A change, and therefore, the temperature-sensitive phenotype may be an effect of two changes. Last, although mutations in the helicase domain render cells sensitive to MMS, most mutations in the nuclease domain do not confer MMS sensitivity. Nevertheless, the dna2-1
mutant is MMS-sensitive, even at the permissive temperature for growth, suggesting effects on the helicase, as well as the nuclease domain.
On all of the substrates tested, WT Dna2 forms two major distinct protein/DNA complexes, whereas the Fe–S mutant proteins show a single complex, corresponding to the most highly retarded and predominant WT Dna2/DNA complex ( and Supplementary Figure S2
). We propose that the faster migrating complex contains a lower molar ratio of Dna2/DNA, probably 1:1, than the slower Dna2/DNA complex, probably 2:1. We do not know whether more than one molecule of Dna2 binds autonomously to the substrate, or whether Dna2 dimerizes through protein/protein interaction, with possibly only one component actually bound to the substrate. The latter is suggested by the appearance of protein/DNA complexes in similar ratios, regardless of ssDNA length (34
). for publication). We also have considered that Dna2 exists in two different forms in cells, each with a specialized function, one would be chelated with a metal ion and one not. In support of this concept, the nuclease activity on the ssDNA substrates was compromised less on flap substrates than in the C to A mutants (). This observation clearly indicates that the metal cluster defective enzyme can still have substantial nuclease activity for ssDNA substrates while lacking the ability to act on other configurations, suggestive that both metal-containing and metal-free enzyme have distinct roles in vivo
. Indeed, we observed two species of Dna2 protein in our original analysis of the purified protein by gel filtration (15
), which might correspond to such species (or to different multimeric species). Possibly, the metal-containing Dna2 is primarily assigned for the 5′- to 3′ nuclease activities in vivo
, whereas Dna2 without metal prefers to show 3′- to 5′ nuclease activity.
An important outcome of this work is that we were able to show that the Dna2 Fe–S cluster has a role in vivo. The expression of WT enzyme, but not any of the cluster mutant enzymes, suppresses the temperature sensitivity of the dna2-1 strain of yeast at 37°C (). Expression of C519A and C768A was able to support growth in the complete absence of Dna2, but C771A and C777A were defective at both 30 and 37°C. This pattern of defective in vivo function mirrors the degree of defect in the nuclease activities in vitro, lending significance to the results. Another interesting observation is that, at permissive temperatures, the mutants are dominant negative and either outcompete or synergistically inactivate the dna2-1 protein.
The presence of an essential Fe–S cluster in Dna2 also has implications related to the observation that Dna2 is localized to the mitochondrion in organisms from yeast to man (35–38
). Formation of Fe–S clusters is not a spontaneous process; a complex biosynthetic assembly machinery is required (29
). Since mitochondria are critical for the synthesis of Fe–S clusters, we have to consider that Dna2 may also localize to the mitochondrion primarily to load Fe in its metal cluster. Furthermore, the efficiency of metal loading is not perfect for any protein. This additionally supports the idea that there are two pools of Dna2 in cells, one loaded with metal and one without.
Recently, it has been reported that the nucleotide excision repair and transcription helicase, XPD; the MutY glycosylase; EndoIII and the SoxR transcription factor, all Fe–S proteins, exhibit ATP-stimulated and DNA-mediated charge transport (40–42
). It has been proposed that this redox activity is important for enzymatic and physiological activity and constitutes a new signaling mechanism in DNA-binding proteins. Investigating the role of the Dna2 Fe–S cluster in potential DNA-mediated charge transport will therefore be interesting.