IncreasedRrm2gene dosage reduces fragile site breakage and prolongs survival of ATR mutant mice

Abstract

In Saccharomyces cerevisiae, absence of the checkpoint kinase Mec1 (ATR) is viable upon mutations that increase the activity of the ribonucleotide reductase (RNR) complex. Whether this pathway is conserved in mammals remains unknown. Here we show that cells from mice carrying extra alleles of the RNR regulatory subunit RRM2 (Rrm2 TG ) present supraphysiological RNR activity and reduced chromosomal breakage at fragile sites. Moreover, increased Rrm2 gene dosage significantly extends the life span of ATR mutant mice. Our study reveals the first genetic condition in mammals that reduces fragile site expression and alleviates the severity of a progeroid disease by increasing RNR activity.Supplemental material is available for this article.Received December 5, 2014; revised version accepted March 2, 2015.Replication stress (RS) has emerged as a source of genome instability in human diseases, including cancer and premature aging (Lecona and Fernandez-Capetillo 2014;Zeman and Cimprich 2014). In brief, RS is defined by the accumulation of abnormal amounts of ssDNA at stalled replication forks that, due to its recombinogenic nature, can initiate genomic rearrangements. In mammals, RS is sensed and suppressed by a signaling cascade initiated by the ATR kinase, which, together with its target kinase, CHK1, suppresses RS through the phosphorylation of multiple targets (Cimprich and Cortez 2008;Lopez-Contreras and Fernandez-Capetillo 2010). ATR and CHK1 are essential for embryonic development in mice (Brown and Baltimore 2000;de Klein et al. 2000;Liu et al. 2000), which is due to the role of the RS response (RSR) in preventing replication-born chromosome breakage. Whether the RSR protects all forks or a subset of them during replication is unclear. On the one hand, proteomic studies of the human replisome in unchallenged conditions have failed to detect ATR or CHK1 in the vicinity of replication forks Sirbu et al. 2013), suggesting that their activity might be particularly necessary for only a subset of forks, such as damaged ones. Accordingly, chromosomal breaks that arise upon ATR inactivation locate preferentially at specific loci named common fragile sites (CFSs) (Casper et al. 2002) and early replicating fragile sites (ERFSs) (Barlow et al. 2013). Regardless of whether ATR works at all forks or only some of them, how it suppresses RS and why it is essential are still not fully understood.Ribonucleotide reductase (RNR) is a tetrameric enzyme composed of two catalytic (RRM1, Rnr1 in yeast) and two regulatory (RRM2, Rnr2 in yeast) subunits (Jordan and Reichard 1998). It reduces NDPs into dNDPs, which is a rate-limiting step for the production of dNTPs. In yeast, the lethality of mec1Δ strains can be bypassed by mutations that increase RNR activity. The first evidence of a connection between ATR and RNR came from the discovery of Crt1 (a transcriptional repressor of RNR subunits) as a suppressor of Mec1 lethality in Saccharomyces cerevisiae (Huang et al. 1998). Furthermore, overproduction of Rnr1 was shown to be sufficient ...

Distinct but Concerted Roles of ATR, DNA-PK, and Chk1 in Countering Replication Stress during S Phase

“…Through these two distinct effects, ATR promotes the synthesis of dNTPs and limits their consumption in early S phase, thereby suppressing ssDNA accumulation and replication catastrophe. Consistent with our findings, a recent study reported that overexpression of RRM2 suppressed the genomic instability in ATR-deficient mice (Lopez-Contreras et al, 2015). Similar to human ATR, the budding yeast ATR homolog Mec1 also promotes dNTP synthesis and prevents inappropriate firing of dormant origins (Huang and Elledge, 1997; Santocanale et al, 1999).…”

Heterozygous colon cancer-associated mutations of SAMHD1 have functional significance

“…Second, inactivation of Sml1, an inhibitor of yeast RNR, leads to a ∼2.5-fold increase in dNTP pools (59), and this slight increase is enough to rescue the lethality of the deletion of MEC1 (yeast homolog of mammalian ATR) (59) and to increase the speed of replication forks by approximately 2-fold (60). Third, in mice, increased gene dosage of the small RNR subunit Rrm2 elevates RNR activity, but does not lead to elevated dNTP pools (61), presumably because of the strict allosteric dATP feedback inhibition of the mammalian RNR. Interestingly, despite any detectable increase in dNTP levels, increased gene dosage of the small RNR subunit Rrm2 reduces fragile site breakage and prolongs the survival of ATR mutant mice (61).…”

“…Third, in mice, increased gene dosage of the small RNR subunit Rrm2 elevates RNR activity, but does not lead to elevated dNTP pools (61), presumably because of the strict allosteric dATP feedback inhibition of the mammalian RNR. Interestingly, despite any detectable increase in dNTP levels, increased gene dosage of the small RNR subunit Rrm2 reduces fragile site breakage and prolongs the survival of ATR mutant mice (61). Fourth, the deletion of Dun1, a protein kinase that controls yeast RNR, causes a ∼50% reduction of dNTP pools that, in turn, decreases the mutagenic effect of proofreading-deficient Pol e (pol2-4) by approximately threefold and results in a mutation rate comparable to WT levels (18).…”

Checkpoint Kinase Rad53 Couples Leading- and Lagging-Strand DNA Synthesis under Replication Stress

“…Genetic evidence indicates that the essential function of Rad53 and Mec1 is linked to their role at DNA replication forks (Desany et al, 1998). Moreover, the essential function of Rad53 and Mec1 (as well as its counterpart ATR) can be suppressed by elevated levels of dNTPs including deletion of SML1 in yeast cells (Lopez-Contreras et al, 2015; Zhao et al, 1998). All these functions of DNA replication checkpoint kinases in the regulation of firing of late-replication origins, up-regulation of dNTP synthesis and maintenance of replisome functions are conserved in human cells.…”