Identification and characterization of CRT10 as a novel regulator of Saccharomyces cerevisiae ribonucleotide reductase genes

Abstract

The CRT10 gene was identified through screening of the Saccharomyces cerevisiae deletion library for hydroxyurea (HU) resistance. CRT10 encodes a putative 957 amino acid, 110 kDa protein with a leucine repeat and a WD40 repeat near the N-terminus. Deletion of CRT10 resulted in an enhanced resistance to HU reminiscent of the inactivation of two other ribonucleotide reductase (Rnr) suppressors, CRT1 and SML1, which regulate Rnr activity at transcriptional and translational levels, respectively. Epistatic analysis indicates that CRT10 belongs to the CRT1 pathway but not the SML1 pathway. Indeed, deletion of CRT10 enhanced the survival of the mec1 null mutant and increased basal level and DNA damage-induced expression of RNR2 and RNR3, suggesting that Crt10 regulates RNR genes at the transcriptional level. Furthermore, the dun1 mutation is epistatic to crt10 with respect to both HU sensitivity and RNR gene expression. Interestingly, the expression of CRT10 itself is induced by DNA damaging agents and this induction requires DUN1, suggesting that CRT10 plays a role in cellular response to DNA damage and replication blocks. The CRT10 function appears to be achieved by positive regulation of the CRT1 transcript level, indicating that CRT10 is a component of the regulatory circuit.

Figure 1

The deduced S.cerevisiae Crt10/YOL063c amino acid sequence. The putative leucine repeat (underlined), transmembrane domain (boldface) and WD repeat (boldface and italicized) are indicated.

Figure 2

Genetic interaction of CRT10 with CRT1 (A), SML1 (B) and MEC1 (C). (A and B) Epistasis analysis. 10-fold serial dilutions of log-phase cultures were spotted on YPD plates and on YPD plates containing HU as described. Plates were incubated for 3 days at 30°C before photographing. Strains used: (A) BY4741 (WT), WX1152 (crt10Δ), WX1153 (crt1Δ) and WXY1154 (crt1Δ crt10Δ). (B) HK578-10A (WT), U952-3B (sml1Δ), WXY1157 (crt10Δ) and WXY1159 (sml1Δ crt10Δ). (C) The inviability of mec1Δ is partially rescued by deletion of CRT10. Tetrads resulting from a cross of WXY1158 (MEC1 SML1 crt10Δ) and U953-61A (mec1Δ sml1Δ CRT10) were dissected and the growth of each spore was followed by microscopic analysis. The representative picture was taken after 4 day incubation at 30°C.

Figure 3

RNR gene expression in wild-type and crt10Δ cells. RNR3-lacZ (A and B) and RNR2-lacZ (C and D) expression was monitored after MMS (A,C) and HU (B,D) treatments. β-gal activity was determined as described in Materials and Methods. (filled square) BY4741 (wild type) and (filled circle) WXY1152 (crt10Δ) were transformed with either pZZ2 (pRNR3-lacZ) or pZZ18 (pRNR2-lacZ) and several independent transformants were picked for analysis. The results are the average of at least three independent experiments with standard deviations. β-gal activity is given in Miller units.

Figure 4

DUN1 is epistatic to CRT10. (A) Deletion of DUN1 abolishes the HU resistance caused by the crt10 mutation. 10-fold serial dilutions were spotted on YPD plates and on YPD plates containing 0.1 M HU. Plates were incubated for 3 days at 30°C before photographing. Strains used: BY4741 (WT), WXY1152 (crt10Δ), WXY1155 (dun1Δ), WX1156 (crt10Δ dun1Δ). (B) The CRT10 effect on RNR3 expression is dependent on DUN1. RNR3-lacZ expression was monitored with or without MMS treatment and expressed in Miller units. The results are the average of at least three independent experiments with standard deviations.

Figure 5

(A) CRT10 expression in response to DNA damage and HU treatment. Log-phase wild-type HK578-10A cells were either untreated (lane 1) or treated with 0.3% MMS for 2 h (lane 2), 0.2 M HU for 2 h (lane 3) or exposed to 40 krad of γ radiation (lane 4). (B) CRT10 induction is DUN1-dependent. Log-phase wild-type BY4741 and its derivatives WXY1153 (crt1Δ) and WXY1155 (dun1Δ) were either untreated (−) or treated with 0.1% MMS for 2 h (+). Northern hybridization was performed as described in Materials and Methods. The membranes were hybridized with CRT10 (upper panel), stripped and then hybridized with ACT1 (lower panel) as an internal control. Each lane contains 15 μg of total RNA.

Figure 6

A proposed model for CRT10 in Rnr regulation. Note that the CRT10 functions and regulation are based on its genetic data instead of protein activity. Note that Rnr catalyzes the rate-limiting step in dNTP production.