Inviability of a DNA2 deletion mutant is due to the DNA damage checkpoint

Abstract

Dna2 is a dual polarity exo/endonuclease, and 5' to 3' DNA helicase involved in Okazaki Fragment Processing (OFP) and Double-Strand Break (DSB) Repair. In yeast, DNA2 is an essential gene, as expected for a DNA replication protein. Suppression of the lethality of dna2Δ mutants has been found to occur by two mechanisms: overexpression of RAD27 (scFEN1) , encoding a 5' to 3' exo/endo nuclease that processes Okazaki fragments (OFs) for ligation, or deletion of PIF1, a 5' to 3' helicase involved in mitochondrial recombination, telomerase inhibition and OFP. Mapping of a novel, spontaneously arising suppressor of dna2Δ now reveals that mutation of rad9 and double mutation of rad9 mrc1 can also suppress the lethality of dna2Δ mutants. Interaction of dna2Δ and DNA damage checkpoint mutations provides insight as to why dna2Δ is lethal but rad27Δ is not, even though evidence shows that Rad27 (ScFEN1) processes most of the Okazaki fragments, while Dna2 processes only a subset.

Figure 1

Mutation of RAD9 suppresses the lethality of dna2Δ. Tetrads from a dissection of MB221 DNA2/dna2Δ::kanMX RAD9/rad9-320 illustrate the presence of a mutation suppressing the lethality of dna2Δ. The dna2Δ colonies are circled.

Figure 2

Expression of RAD9 in a dna2Δ rad9-320 strain results in lethality. (A) Contol showing that RAD9 expression in DNA2 rad9Δ is not lethal. Strain MB228-DNA2::9MYC rad9Δ::HIS3 was transformed with the plasmids pSEY-18GAL and BG1805-Rad9. Cells carrying the plasmids were incubated on medium lacking uracil containing 2% glucose, 0.05% galactose 2% raffinose or 0.2% galactose 2% raffinose. (B) Expression of RAD9 in dna2Δ rad9-320 is lethal. Strain MB220 dna2Δ rad9-320 was trainsformed with the plasmids pSEY18GAL, BG1805-Rad9 and pSEY18GAL-DNA2 and incubated with 2% glucose, 0.05% galactose 2% raffinose, 0.2% galactose 2% raffinose.

Figure 3

rad9Δ mrc1AQ double mutation suppresses the temperature-sensitive growth of dna2-1 mutants. Mutants dna2-1, dna2-1 rad9Δ, dna2-1 mrc1AQ, dna2-1 rad9Δ mrc1AQ, rad9Δ mrc1AQ were incubated on plates at 23°C, 28°C and 34°C.

Figure 4

rad9Δ mrc1AQ double mutation suppresses the lethality of dna2Δ better than rad9Δ alone. Tetrads from a dissection of MB227 DNA2/dna2Δ::natR MRC1/mrc1AQ::HIS3 RAD9/rad9Δ::kanMX. The dna2Δ rad9Δ colonies are enclosed by a square, and dna2Δ rad9Δ mrc1AQ colonies are enclosed by a circle.

Figure 5

Checkpoint activation due to replication errors in the absence of Dna2. In vitro, DNA strand displacement synthesis by pol δ in the presence of Rad27^scFEN1 protein on a primer adjacent to an upstream fragment results in the appearance of cleaved flaps. Although most of the cleaved flaps are short, 8–12 nt, a small subset are about 30 nt., Pol δ strand displacement is stimulated by Pif1, and a significant increase in the 30 bp cleaved product is observed in the presence of Pif1, although the 30 nt cleaved product is still only 1–2% of the total cleaved products., The 30 nt flap synthesized by Polδ/Pif1 becomes refractory to cleavage by Rad27^scFEN1 in the presence of RPA, which may result in even further lengthening of the flap., As shown in (A), in normal cells containing Dna2, the RPA bound flap is bound by Dna2, which cleaves the long flap, releasing RPA and allowing Rad27^scFEN1 to further cleave the flap, resulting in a ligatable nick.–,, In the absence of Dna2, (B), the RPA-bound flap, however, persists and can bind Mec1/Ddc2 kinase. The 5′ flap-bound Mec1/Ddc2 then recruits and phosphorylates Rad9, which binds methylated histone H3-K79 with its Tudor domain. Mec1/Ddc2 may also phosphorylate histone H2A, providing an additional site for Rad9 binding to chromatin. Chromatin bound Rad9 then recruits Rad53, resulting in dimerization, autophosphorylation and Rad53 kinase activation, allowing Rad53 to activate the DNA damage response and cell cycle arrest. (Similarly, the MCM helicase and pol ε may target Mrc1 to Mec1-bound 5′ flaps). After checkpoint activation, cells normally repair the DNA damage, deactivate the checkpoint and resume cell division. dna2Δ strains would be unable to repair the unprocessed Okazaki fragments during the G₂ division delay; thus the Rad9/Mrc1-dependent G₂ division delay contributes to cell death rather than recovery.