Interplay of Mre11 nuclease with Dna2 plus Sgs1 in Rad51-dependent recombinational repair

Abstract

The Mre11/Rad50/Xrs2 complex initiates IR repair by binding to the end of a double-strand break, resulting in 5' to 3' exonuclease degradation creating a single-stranded 3' overhang competent for strand invasion into the unbroken chromosome. The nuclease(s) involved are not well understood. Mre11 encodes a nuclease, but it has 3' to 5', rather than 5' to 3' activity. Furthermore, mutations that inactivate only the nuclease activity of Mre11 but not its other repair functions, mre11-D56N and mre11-H125N, are resistant to IR. This suggests that another nuclease can catalyze 5' to 3' degradation. One candidate nuclease that has not been tested to date because it is encoded by an essential gene is the Dna2 helicase/nuclease. We recently reported the ability to suppress the lethality of a dna2Delta with a pif1Delta. The dna2Delta pif1Delta mutant is IR-resistant. We have determined that dna2Delta pif1Delta mre11-D56N and dna2Delta pif1Delta mre11-H125N strains are equally as sensitive to IR as mre11Delta strains, suggesting that in the absence of Dna2, Mre11 nuclease carries out repair. The dna2Delta pif1Delta mre11-D56N triple mutant is complemented by plasmids expressing Mre11, Dna2 or dna2K1080E, a mutant with defective helicase and functional nuclease, demonstrating that the nuclease of Dna2 compensates for the absence of Mre11 nuclease in IR repair, presumably in 5' to 3' degradation at DSB ends. We further show that sgs1Delta mre11-H125N, but not sgs1Delta, is very sensitive to IR, implicating the Sgs1 helicase in the Dna2-mediated pathway.

Figure 1. X-ray resistance of the dna2Δ pif1Δ strain compared to sensitivity of mre11Δ pif1Δ.

The strains MB120-5A (WT), MB121- pif1Δ, MB161B- dna2Δ pif1Δ, MB122-17C- mre11Δ, MB124-2D- mre11Δ pif1Δ were harvested in mid-log phase, resuspended in water, irradiated, serially diluted (1∶10), and plated.

Figure 2. dna2Δ pif1Δ mre11Δ (pRS414mre11-D56N) and dna2Δ pif1Δ mre11Δ (pRS414mre11-H125N) strains exhibit similar sensitivity to X-rays as mre11Δ pif1Δ.

These strains were created by dissecting tetrads from the following crosses: MATa dna2Δ pif1Δ trp1Δ bar1Δ/ MATα mre11Δ trp1Δ (pRS414MRE11), MATa dna2Δ pif1Δ trp1Δ bar1Δ/ MATα mre11Δ trp1Δ (pRS414mre11-D56N) and MATa dna2Δ pif1Δ trp1Δ bar1Δ/ MATα mre11Δ trp1Δ (pRS414mre11-H125N) diploids. Trp⁺ spores were replica plated, and irradiated. Control strains mre11Δ pif1Δ (pRS414MRE11) and dna2Δ pif1Δ mre11Δ (pRS414MRE11) were identified from the respective cross and were X-ray resistant. dna2Δ pif1Δ mre11Δ (pRS414mre11D56N) or dna2Δ pif1Δ mre11Δ (pRS414mre11H125N), from the two respective crosses were radiation sensitive. To compare the radiation sensitivity of the dna2Δ pif1Δ mre11Δ (pRS414mre11-D56N) and the dna2Δ pif1Δ mre11Δ (pRS414mre11-H125N) (bottom panel) to mre11Δ pif1Δ (pRS414mre11-D56N) and to mre11Δ pif1Δ (pRS414mre11-H125N) strains (top panel), serially diluted cells were irradiated with 26 krads on a plate and allowed to grow for three days. Note that a dna2Δ pif1Δ mre11Δ strain carrying only the pRS vector is inviable, so an mre11Δ pif1Δ strain carrying the pRS vector alone is shown as the control, as indicated by the * in the bottom panel.

Figure 3

(A) X-ray sensitivity of a dna2Δ pif1Δ mre11-D56N strain and complementation by CEN plasmids containing Mre11 or Dna2. DNA fragments containing mre11-D56N and mre11-H125N mutant genes with 800 bp 5′ and 140 bp 3′ to the gene were cloned into pRS306URA3, cleaved in the mre11 promoter and transformed into a dna2Δ pif1Δ MRE11 strain to allow integration at the MRE11 locus. The resulting transformants were propagated on YPD media then grown on 5-FOA containing media to excise the MRE11 allele. Approximately 15% of the pRS306mre11-D56N and pRS306mre11-H125N Ura⁻ pop outs were X-ray sensitive. Strains MB161B-56- dna2Δ pif1Δ mre11D56N, MB161B-56 (pRS414MRE11), MB161B (pRS314DNA2) were then compared for X-ray sensitivity as in Figure 1. (B) X-ray sensitivity of a dna2Δ pif1Δ mre11-H125N strain and complementation by plasmids containing MRE11 or nuclease proficient DNA2 genes. Strain MB161B-125 – dna2Δ pif1Δ mre11H125N, MB161B-125 (pRS414MRE11), MB161B-125 (pR314DNA2), MB161B (pRS314dna2K1080E), MB161B-125 (pRS314dna2-2) were compared for X-ray sensitivity as described in Figure 1.

Figure 4

(A) Comparison of the X-ray sensitivity of a mre11-D56N pif1Δ strain with a dna2Δ mre11-D56N pif1Δ strain. The integrating plasmids pRS306mre11-D56N::URA3 and pRS306mre11-H125N::URA3 were cut with Sph1 which cleaves about 300 bp 5′ to the ATG of MRE11 and transformed into a MATa mre11Δ::natR strain and insertion at the MRE11 locus was selected for on G418 plates lacking uracil. These strains were mated with MB161B-MATa dna2Δ pif1Δ MRE11, and the diploids were sporulated and dissected. When the resulting spores were scored, the NatR gene always segregated with the URA3 gene, demonstrating that the pRS306mre11nuclease minus plasmids were integrated at the MRE11 loci in the respective strains. Strains MB126-mre11Δ::natR::mre11D56N::URA3, MB128- mre11Δ::natR::mre11-D56N::URA3 pif1Δ, and MB133 - dna2Δ pif1Δ mre11Δ::natR::mre11-D56N::URA3 were treated as in Figure 1 to determine IR sensitivity. (B) Comparison of the X-ray sensitivity of a mre11-H125N pif1Δ strain with a dna2Δ mre11-H125N pif1Δ strain. Strains were constructed as in Figure 4A. Strains MB127-mre11Δ::natR::mre11-H125N::URA3, MB129-mre11Δ::natR::mre11-H125N::URA3 pif1Δ, and MB134 - dna2Δ mre11Δ::natR::mre11-H125N::URA3 pif1Δ, were treated as in Figure 1.

Figure 5. Comparison of the X-ray sensitivity of strain sgs1Δ with sgs1Δ mre11-H125N and sgs1Δ sae2Δ.

Strains MB137 sgs1Δ, MB138 sgs1Δ mre11-H125N and MB135 sae2Δ and MB136 sgs1Δ sae2Δ were constructed in this study and treated as in Figure 1.

Figure 6. Expression of nuclease deficient Dna2 is toxic in a dna2Δ pif1Δ strain.

Strain MB21-dna2Δ pif1Δ carrying either pRS314DNA2, Hel⁺, Nuc⁺; pRS314, Vector; pRS314dna2K1080E, Hel⁻Nuc⁺; pRS314dna2E675A, Hel⁺Nuc⁻ or pRS314dna2K1080E,dna2E675A, Hel⁻Nuc⁻ plasmids was grown, serially diluted 10 fold and plated on trptophan deficient plates with or without 26 krads IR, and allowed to grow for four days at the temperatures indicated.

Figure 7. Inactivation of the Dna2 nuclease is lethal in a pif1Δ mre11-H125N strain.

A dna2Δ pif1Δ mre11-H125N trp1Δ (pGAL::DNA2URA3) strain was transformed with the Trp⁺ plasmids pRS314DNA2, Hel⁺, Nuc⁺; pRS314, Vector; pRS314dna2K1080E, Hel⁻Nuc⁺; pRS314dna2E675A, Hel⁺Nuc⁻ or pRS314dna2K1080E,dna2E675A, Hel⁻Nuc⁻. The Trp⁺Ura⁺ transformants were grown in tryptophan deficient media and spotted onto tryptophan minus (left) or tryptophan minus, 5-FOA containing (right) media at 23°C and photographed after 4 days at 23°C.