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. 2002 Mar 1;30(5):1224-32.
doi: 10.1093/nar/30.5.1224.

Homologous recombination is essential for RAD51 up-regulation in Saccharomyces cerevisiae following DNA crosslinking damage

Yuval Cohen  1 , Michele Dardalhon, Dietrich Averbeck
Affiliations

Homologous recombination is essential for RAD51 up-regulation in Saccharomyces cerevisiae following DNA crosslinking damage

Yuval Cohen et al. Nucleic Acids Res. .

Abstract

We have determined the kinetics of up-regulation of the homologous recombination gene RAD51, one of the genes induced following DNA damage in isogenic haploid DNA repair-deficient mutants of Saccharomyces cerevisiae, using treatment with the DNA crosslinking agent 8-methoxypsoralen. We show that RAD51 is up-regulated concomitantly, although independently, with a shift from the G1 cell cycle phase to G2/M arrest. This up-regulation is absent in homologous recombination repair-deficient mutants and increased in mutants deficient in nucleotide excision repair and pol(zeta)-dependent translesion synthesis. We demonstrate that the Rad53-dependent DNA damage signal transduction cascade is active in RAD51 non-inducing mutants. However, when independently eliminated, it too abolishes RAD51 up-regulation. We present a model in which RAD51 up-regulation requires two signals: one depending on the Rad53-dependent DNA damage signal transduction cascade and the other on homologous recombination repair.

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Figures

Figure 1
Figure 1
Survival data for isogenic DNA repair-deficient mutants following 8-MOP plus UVA treatment. Cells were grown and treated as described in Materials and Methods.
Figure 2
Figure 2
Responses to 8-MOP damage in G1 synchronized wild-type cells. (A) Northern blot analysis of RAD51 mRNA levels following 8-MOP plus three different UVA doses (1, 2 and 3 kJ/m2). Induction was normalized using actin (ACT1) transcript levels and is given in arbitrary units (see Materials and Methods). (B) Cell cycle changes following damage as monitored by FACS analysis. C1 denotes single content (equivalent to haploid G1) and C2 denotes double content (equivalent to haploid G2). (C) Interferential (DIC) and fluorescence (DAPI staining) light microscopy of treated cells at times 0 and 2 h post-treatment (1 kJ/m2).
Figure 3
Figure 3
Up-regulation of RAD51 in G1 synchronized DNA repair-deficient mutants following 8-MOP plus UVA treatment. (A) Homologous recombination-deficient rad50, rad52, rad54 and rad51-E221K (strain AA106) mutants (2 kJ/m2). (B) NHEJ-deficient yku70 mutant (2 kJ/m2). (C) NER-deficient rad14 mutant (3 kJ/m2). (D) Polζ-dependent TLS-deficient rev3 and post-replication repair-deficient rad18 mutants (1 kJ/m2).
Figure 4
Figure 4
Cell cycle changes following 8-MOP damage. FACS profiles of G1 synchronized DNA repair-deficient mutants following treatment with 8-MOP plus UVA at different UVA doses (indicated). In all cases, the FACS profile for the untreated sample was identical to the wild-type (see Fig. 2B). C1 denotes single content (equivalent to haploid G1) and C2 denotes double content (equivalent to haploid G2).
Figure 5
Figure 5
(A) RAD51 up-regulation in rad14rev3 and rad14rad50 mutants following treatment with 8-MOP plus 1 kJ/m2 UVA. The assay was carried out on asynchronous populations. (B) Induction of CANR mutations after treatment with 8-MOP plus UVA (see Materials and Methods).
Figure 6
Figure 6
(A) Phosphorylation of Rad53 following 8-MOP plus UVA damage in wild-type, rad50, rad52 and rev3 repair-deficient mutants treated with 2 kJ/m2 UVA plus 8-MOP. For the conditions of western blotting see Materials and Methods. (B) RAD51 up-regulation in the mec-sml1 mutant following DNA damage. Northern blot analysis was carried out on asynchronous cells treated with 3 kJ/m2 UVA plus 8-MOP.
Figure 7
Figure 7
A model for up-regulation of RAD51 in 8-MOP-treated cells. The Rad53-dependent DNA damage signal transduction cascade (left) and repair events (right) leading to up-regulation of RAD51. The 8-MOP photolesion (represented as a diagonal bar between the DNA strands) is either not processed or is incompletely processed by NER, leading to a blocked replication fork resulting in a DSB followed by channeling into homologous recombination or TLS. Passage through the homologous recombination pathway generates intermediates that act as a signal for up-regulation of RAD51. The absence of homologous recombination eliminates RAD51 up-regulation but has no effect on Rad53 phosphorylation or cell cycle changes (see Discussion). Lesions processed by TLS do not generate a signal for RAD51 up-regulation. Proteins lacking in relevant mutants are marked in accordance with their respective pathways. At the same time, a signal is transmitted through the Rad53-dependent DNA damage signal transduction cascade, resulting in cell cycle arrest. This signal is necessary, but insufficient, for RAD51 up-regulation. Neither signal can compensate for the other, demonstrating that both are equally needed.
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