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. 2007 Dec;27(23):8409-18.
doi: 10.1128/MCB.01543-07. Epub 2007 Oct 8.

Requirement of Nse1, a subunit of the Smc5-Smc6 complex, for Rad52-dependent postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae

Sergio R Santa Maria  1 , Venkateswarlu Gangavarapu, Robert E Johnson, Louise Prakash, Satya Prakash
Affiliations

Requirement of Nse1, a subunit of the Smc5-Smc6 complex, for Rad52-dependent postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae

Sergio R Santa Maria et al. Mol Cell Biol. 2007 Dec.

Abstract

In Saccharomyces cerevisiae, postreplication repair (PRR) of UV-damaged DNA occurs by a Rad6-Rad18- and an Mms2-Ubc13-Rad5-dependent pathway or by a Rad52-dependent pathway. The Rad5 DNA helicase activity is specialized for promoting replication fork regression and template switching; previously, we suggested a role for the Rad5-dependent PRR pathway when the lesion is located on the leading strand and a role for the Rad52 pathway when the lesion is located on the lagging strand. In this study, we present evidence for the requirement of Nse1, a subunit of the Smc5-Smc6 complex, in Rad52-dependent PRR, and our genetic analyses suggest a role for the Nse1 and Mms21 E3 ligase activities associated with this complex in this repair mode. We discuss the possible ways by which the Smc5-Smc6 complex, including its associated ubiquitin ligase and SUMO ligase activities, might contribute to the Rad52-dependent nonrecombinational and recombinational modes of PRR.

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Figures

FIG. 1.
FIG. 1.
Isolation of the nse1-101 mutant of S. cerevisiae. (A) Sequence alignment of the C termini of Nse1 proteins from various organisms. Multiple-sequence alignment was performed with CLUSTAL W version 1.82. Sequences aligned are S. cerevisiae Nse1 (ScNse1; 336 amino acids; GenBank accession no. NP_013107), S. pombe Nse1 (SpNse1; 232 amino acids; NP_588097), Homo sapiens Nse1 (HsNse1; 256 amino acids; NP_659547), and Mus musculus Nse1 (MmNse1; 280 amino acids; NP_080606). Shaded residues correspond to the C4HC3 RING zinc finger-like motif. Residues with an asterisk (G175E, S207T, and G332D) are mutated in the nse1-101 mutant. Cysteine 274 (•) was mutated to alanine (C274A) in the nse1 C274A mutant. (B) Photomicrograph of wild-type NSE1 and nse1-101 mutant cells grown at 30°C. (C) Sensitivity of the nse1-101 mutant to DNA-damaging agents. (1) Growth on a YPD plate at 30°C for 2 days. (2) Growth deficiency on a YPD plate at the nonpermissive temperature (37°C). (3) Cells were spotted on a YPD plate, UV irradiated at 80 J/m2, and incubated at 30°C for 2 days. (4) Cells were spotted on a YPD plate containing 0.035% MMS and incubated at 30°C for 2 days.
FIG. 2.
FIG. 2.
Epistasis analysis of the nse1-101 mutation. The nse1-101 mutation enhances the UV sensitivity of the rad14Δ, rad18Δ, rad5Δ, mms2Δ, and ubc13Δ mutants but not of the rad52Δ mutant. The UV sensitivity of the pol30-119 mutant, defective in lysine 164 ubiquitination and the sumoylation of PCNA, is enhanced in the presence of nse1-101. YPD plates containing 10-fold dilutions of exponentially growing yeast cells were UV irradiated at the indicated doses and incubated in the dark for 2 days at 30°C.
FIG. 3.
FIG. 3.
Requirement of the NSE1 gene for PRR of UV-damaged DNA. Sedimentation in alkaline sucrose gradients of nuclear DNA from cells incubated for different periods following UV irradiation. The rad1Δ (A) and rad1Δ nse1-101 (B) strains were UV irradiated at 3.5 J/m2 and then pulse-labeled with [3H]uracil for 15 min, followed by a 30-min chase (▵), a 6-h chase at 30°C (•), or a 6-h chase at 37°C (▪) in high-uracil medium. DNA synthesized in unirradiated cells was pulse-labeled with [3H]uracil for 15 min, which was followed by a 6-h chase (○) at 30°C for the rad1Δ strain and at 37°C for the rad1Δ nse1-101 strain; in unirradiated rad1Δ nse1-101 cells that were treated similarly but kept for 6 h at 30°C, normal-size DNA was reconstituted in a manner similar to that seen in cells held at 37°C. The data for the rad1Δ strain in panel A shown here for comparison are taken from reference .
FIG. 4.
FIG. 4.
Involvement of NSE1 in the RAD52-dependent PRR pathway of UV-damaged DNA. Sedimentation in alkaline sucrose gradients of nuclear DNA from cells incubated for different periods following UV irradiation. The rad1Δ rad5Δ (A), rad1Δ rad52Δ (B), rad1Δ rad5Δ nse1-101 (C), and rad1Δ rad52Δ nse1-101 (D) strains were UV irradiated at 3.5 J/m2 and then pulse-labeled with [3H]uracil for 15 min, followed by a 30-min chase (▵), a 6-h chase at 30°C (•), or a 6-h chase at 37°C (▪) in high-uracil medium. DNA synthesized in unirradiated cells was pulse-labeled with [3H]uracil for 15 min, which was followed by a 6-h chase at 30°C (○) for the rad1Δ rad5Δ and rad1Δ rad52Δ strains and at 37°C for the rad1Δ rad5Δ nse1-101 and rad1Δ rad52Δ nse1-101 strains. The data for the rad1Δ rad5Δ strain in panel A and for the rad1Δ rad52Δ strain in panel B, which are shown here for comparison, are taken from references and , respectively.
FIG. 5.
FIG. 5.
Epistasis analysis of the nse1 C274A mutation. (A) Photomicrograph of nse1 C274A mutant cells showing aberrant morphology compared with that of wild-type cells at 30°C. (B) Sensitivity of the nse1 C274A mutant to DNA-damaging agents. (1) Growth on a YPD plate at 30°C for 2 days. (2) Growth deficiency on a YPD plate at the nonpermissive temperature (37°C). (3) Cells were spotted on a YPD plate, UV irradiated at 80 J/m2, and incubated at 30°C for 2 days. (4) Cells were spotted on a YPD plate containing 0.035% MMS and incubated at 30°C for 2 days. (C) The nse1 C274A mutation enhances the UV sensitivity of the rad14Δ, rad18Δ, rad5Δ, and pol30-119 mutants but has no significant effect on the UV sensitivity of the rad52Δ mutant. YPD plates containing 10-fold dilutions of exponentially growing yeast cells were UV irradiated at the indicated doses and incubated in the dark for 2 days at 30°C.
FIG. 6.
FIG. 6.
Epistasis analysis of the mms21 RING mutation. (A) Sequence alignment of the C-terminal SP-RING domain of Mms21 proteins from various organisms. Multiple-sequence alignment was performed with CLUSTAL W version 1.83. Sequences aligned are S. cerevisiae Mms21 (ScMms21; 267 amino acids; GenBank accession no. NP010896), S. pombe Mms21 (SpMms21; 250 amino acids; Q4PIR3), Homo sapiens Mms21 (HsMms21; 247 amino acids; NP775956), and Mus musculus Mms21 (MmMms21; 247 amino acids; NP081022). Shaded residues correspond to the zinc-binding residues of the SP-RING domain. Residues marked with an asterisk (C200A and H202A) are mutated in the mms21 RING mutant. (B) Photomicrograph of mms21 RING mutant cells showing aberrant morphology compared to that of wild-type cells at 30°C. (C) Sensitivity of the mms21 RING mutant to DNA-damaging agents. (1) Growth on a YPD plate at 30°C for 2 days. (2) Growth deficiency on a YPD plate at the nonpermissive temperature (37°C). (3) Cells were spotted on a YPD plate, UV irradiated at 80 J/m2, and incubated at 30°C for 2 days. (4) Cells were spotted on a YPD plate containing 0.035% MMS and incubated at 30°C for 2 days. (D) The mms21 RING mutations enhance the UV sensitivity of the rad14Δ, rad18Δ, and pol30-119 mutants but have no significant effect on the UV sensitivity of the rad52Δ mutant. YPD plates containing 10-fold dilutions of exponentially growing yeast cells were UV irradiated at the indicated doses and incubated in the dark for 2 days at 30°C.
FIG. 7.
FIG. 7.
Rad6-Rad18-dependent and Smc5-Smc6/Nse1-Mms21-dependent pathways for the replication of UV-damaged DNA in yeast. (A) Rad6-Rad18-dependent replication through UV lesions can occur either by TLS mediated by Polη or Polζ or by Rad5-mediated fork regression and template switching. The Smc5-Smc6 complex functions in the Rad52-dependent PRR pathway, which is proposed to act in a nonrecombinational manner. (B) In the K164R PCNA mutant, because of the absence of PCNA sumoylation, the Rad52 recombinational pathway becomes activated and the Smc5-Smc6 complex acts in this pathway also (for more details, see Discussion).
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