In this work we examined minisatellite alterations during stationary phase in S. cerevisiae. First, we demonstrated that ZRT1-dependent stationary phase minisatellite alterations occur specifically in quiescent cells, and not in nonquiescent cells within the stationary phase population. We showed that minisatellite alterations in Δzrt1 mutants are not dependent on chromosomal context or adenine auxotrophy. We determined that recombination factors are required for alterations in minisatellite tracts during quiescence. The majority of minisatellite alterations in a Δzrt1 strain occurs by RAD52-dependent homologous recombination or a RAD52-independent pathway that requires RAD50. Finally, we find that mutation of ZRT1 can destabilize a human minisatellite, implying that zinc homeostasis may play a role in minisatellite stability in human cells.
Stationary phase cultures of S. cerevisiae
consist of a quiescent cell fraction, in which cells are uniformly arrested and bulk DNA synthesis does not occur, and a nonquiescent cell fraction, in which some budded cells are present and DNA replication may occur [28
]. Therefore, we considered that ZRT1
-dependent minisatellite alterations during stationary phase might occur as a result of polymerase slippage during whole-genome DNA synthesis in nonquiescent cells. However, we found that loss of ETR1
, which specifically reduces the ability of quiescent cells to reenter the cell cycle, completely eliminated blebbing in a Δzrt1
mutant. In contrast, loss of POR1
, which specifically reduces the reproductive capacity of nonquiescent cells, has little effect on blebbing in a Δzrt1
mutant. Therefore, our data argue that ZRT1
-dependent minisatellite alterations occur as a result of events in quiescent cells that do not require bulk DNA synthesis. Recent work [38
] demonstrates limited DNA synthesis at specific locations in the genome of stationary phase yeast cells; the events that lead to minisatellite tract alterations may utilize this type of limited repair synthesis.
Using the chromosomal ade2-min3 allele (), we determined that loss of ZRT1 triggers an increase in minisatellite alterations exclusively in quiescent cells. We utilized the ura3-min3 allele to show that minisatellite alterations in a Δzrt1 mutant strain occur independently of chromosomal context and adenine auxotrophy. In stationary phase Δzrt1 cells, we see an increase in plasmid-borne ura3-min3 minisatellite alterations, eliminating the possibility that the Δzrt1 minisatellite instability phenotype is an artifact of cis-acting sequences surrounding ADE2 and implying that chromosomal context is not likely to have an influence on these events. Alterations occurred with equal frequency in Ade+ or Ade− strains, indicating that the alterations are not due to the disruption of the adenine biosynthetic pathway. Taken together, these results argue that loss of ZRT1 could potentially destabilize minisatellites at many loci and in many genetic backgrounds.
Our data demonstrate that most minisatellite alterations in a Δzrt1 mutant require homologous recombination. Loss of RAD50, RAD51, or RAD52 in a Δzrt1 mutant strain reduces the minisatellite alterations by approximately 50% compared to the parental Δzrt1 single mutant (). Similarly, loss of both RAD50 and RAD51 or both RAD51 and RAD52 in a Δzrt1 mutant also reduce minisatellite alterations by ~50%. However, loss of both RAD50 and RAD52 in a Δzrt1 strain reduces minisatellite alterations by ~81%, indicating that both RAD52-dependent recombination and a RAD50-dependent, RAD52-independent mechanism are required for minisatellite alterations in the Δzrt1 mutant. Finally, loss of RAD50, RAD51, and RAD52 in a Δzrt1 mutant reduces minisatellite alterations to a level not significantly different from the parental Δrad50 Δrad51 Δrad52 strain, indicating RAD51 plays a role in ZRT1-dependent minisatellite alterations in the absence of RAD50 and RAD52. Finally, deletion of the non-homologous end-joining (NHEJ) ligase DNL4 in a Δzrt1 mutant strain does not reduce the frequency of minisatellite alterations, but a significant reduction is seen when RAD52 is also deleted, indicating that NHEJ can play a role in stability maintenance if the homologous recombination pathway has been compromised. These results are consistent with a model in which two homologous recombination pathways, one RAD52-dependent and the other RAD52-independent and requiring RAD50, are required for most alterations occurring in the Δzrt1 mutant cells, with other pathways becoming active when homologous recombination has been compromised.
While recombinational mechanisms have previously been implicated in minisatellite instability during yeast mitosis [1
], minisatellite alterations in Δzrt1
mutants occur during stationary phase, a stage at which most yeast cells are quiescent [39
]. Recombination modulation previously has been linked to genome alterations during stationary phase in prokaryotic E. coli
cells (see, for example [40
]); our data extend these findings to eukaryotic yeast cells.
Stationary phase in our Δzrt1
strain consists of a relatively uniform G1 arrest; visual inspection of the ade2-min3 Δzrt1
strain shows that by the time the culture enters stationary phase, which occurs at ~48hrs after inoculation [22
], approximately 98% of the cells are unbudded (G1) (data not shown). G1 cells have only one copy of each chromosome while G2 cells, which have undergone DNA replication, have two sister chromatids. Thus, minisatellite alterations in a Δzrt1
mutant are not likely to be a result of recombination between sister chromatids. This is significant because all the strains we have used to examine minisatellite stability in this study are haploid and therefore cannot undergo recombination between homologous chromosomes.
What is the molecular mechanism underlying stationary phase minisatellite tract alteration? Since we have shown that ZRT1
-dependent minisatellite alterations occur in quiescent, G1-arrested stationary phase haploid cells, the mechanism for these events must not rely on bulk DNA synthesis or involve exchange between sister chromatids or homologous chromosomes. Therefore, we propose that minisatellite alterations in Δzrt1
mutants during stationary phase occur by two mechanisms: single strand annealing (SSA) and intramolecular repair events. Simple misalignment of repeats during SSA could easily result in minisatellite repeat deletion, and the role of SSA in repeat array contraction has been well established [34
]. An intramolecular repair event could be initiated by a single-strand gap (). During the limited DNA synthesis needed to repair the gap, polymerase slippage and misalignment could result in repeat units forming a single-stranded loop. If the loop forms on the template DNA strand and is removed, a deletion of repeat units will result. If the loop forms on the newly synthesized DNA strand and is repaired by nicking the template DNA strand opposite the loop, an expansion of the repeat tract will result (). The frequency of this type of event is dependent on the frequency of polymerase slippage and the formation of single-stranded nicks or gaps. It is quite possible that the polymerase slippage frequency is significantly increased at repetitive tracts in stationary phase cells. Nucleotide reserves in quiescent cells are likely to be small relative to the nucleotide pools available in actively-dividing cells, leading to increased polymerase pausing (especially at repetitive DNA tracts) thereby increasing the likelihood of polymerase dissociation and re-association events. Finally, defects in zinc transport have been shown to elevate single-stranded DNA damage in mammalian cells [43
A model for intramolecular minisatellite alterations.
Our data on the types of minisatellite alterations observed in Δzrt1
mutants are consistent with these models. We previously showed that minisatellite alterations in Δzrt1
mutants consist of deletions of repeat units from the ade2-min3
]. In this study we observed both repeat deletions and tract expansion in the ade2-h7.5
minisatellite (). Intramolecular repair events and SSA can both lead to deletions in direct repeat tracts when repeats are misaligned [34
]. We find that minisatellite alterations in a Δzrt1
mutant during stationary phase occur by both RAD52
-dependent and RAD52
-independent mechanisms. SSA is a RAD52
-dependent process for shorter repeat arrays [45
-independent mechanisms of SSA have been demonstrated only for large CUP1
and rDNA repeat tracts [47
]. Since our ade2-min3
reporters are significantly smaller than those tracts both in terms of repeat unit length and number of repeats, it is unlikely that RAD52
-independent SSA contributes to our events. RAD59
are involved in SSA [36
]. Deletion of either RAD1
in a Δzrt1
strain leads to a ~20% reduction in minisatellite alterations (, data not shown), clearly implicating SSA in these events. Since some SSA has been demonstrated to occur even without RAD59
], it is difficult to estimate what proportion of ZRT1
-dependent minisatellite alterations result from SSA. It may be that all RAD52
-dependent minisatellite alterations in the Δzrt1
mutant occur by SSA. However, as we have argued above, RAD52-
independent SSA is not likely to contribute to minisatellite alterations in the ade2-min3
minisatellite tract. Therefore, all RAD52
-independent minisatellite alterations in the ZRT1
mutant may occur via some form of intramolecular repair. Consistent with this, we find that most RAD52
-independent minisatellite alterations require RAD50
; prior studies demonstrated a requirement for RAD50
-independent intramolecular recombination [34
]. Rad50p possesses zinc hooks that are employed in linking DNA ends [50
], which could play a role in forming the single stranded DNA loops in our model of intramolecular minisatellite repair. Previous work has demonstrated that single stranded DNA loops over 16 nt can be repaired by loop removal, but the genetic requirements for this process remain elusive, likely because redundant pathways facilitate it [52
]. Therefore, it is difficult to provide evidence for this aspect of our model. Finally, our DNL4
data show that NHEJ can be used as a repair mechanism when homologous recombination has been compromised.
Importantly, we demonstrate here that a human HRAS1
minisatellite tract alters in quiescent cells, and that loss of ZRT1
further destabilizes the minisatellite (). The yeast ZRT1
protein is a member of the ZIP (Zrt-, Irt-like Protein) family of zinc transporters, which are found throughout bacteria and eukaryotes, including humans [53
]. Mammals have three orthologs of the yeast ZRT1
gene: ZIP1 (also known as ZIRTL), ZIP2 and ZIP3 [54
]. The ZIP1 protein in humans (hZip1) is likely to be the major zinc transporter for most of the cells in the body, as it is expressed in most cell types [59
]. The human Zip2 protein is expressed in prostate and uterine epithelial cells. Mutations in ZIP family zinc transporters lead to zinc deficiency and associated health problems in humans [60
]. For example, mutation of Zip2 or Zip3 has been linked to prostate cancers [54
Our data provide a mechanism to link zinc deficiencies with human disease generation. DNA strand breaks, triggered by zinc deficiency, could be the initiating lesions for minisatellite alterations in post-mitotic cells. In agreement with this model, zinc deficiency has been linked to increased DNA strand breakage in mammals [43
]. Variations in the HRAS1
minisatellite tract are known to alter HRAS1
], modifying the activity of this important oncogene and influencing the onset of particular cancers. These alteration events may be elevated in quiescent, post-mitotic cells, as our data demonstrate that they are particularly sensitive to loss of zinc transporters.
Links that support this hypothesis have been detected between zinc homeostasis and minisatellite stability in human disease. Prostate cancer has been linked to both zinc deficiency and rare HRAS1
minisatellite alleles [8
]. Type 1 diabetes has long been associated with rare alleles of the IDDM1
minisatellite, and has recently been correlated with SNPs in the ZIP family zinc transporter SLC30A8 [3
]. Thus, the proposal that zinc homeostasis may directly affect minisatellite stability and influence minisatellite-correlated disease in humans clearly merits further investigation.