We previously reported the identification and characterization of two regions of chromosome III (FS1 and FS2) that are hot spots for chromosome rearrangements in yeast strains with low levels of DNA polymerase α (20
). We argued that these sites were analogous to fragile sites in mammalian cells, which are hot spots for chromosome breakage in cells incubated in drugs that inhibit DNA replication (3
). In this study, we examine the effects of low levels of DNA polymerase δ in several different assays of genome stability: sensitivity to DNA-damaging agents, rate of chromosome loss and mitotic recombination, and rate of illegitimate mating. In many of these assays, reduced levels of DNA polymerase δ and DNA polymerase α have similar effects. Given the quite different roles of the two DNA polymerases at the replication fork and in DNA repair, this result is somewhat surprising.
We found that the reduced expression of Pol3p results in a 12-fold increase in the forward mutation rate of the CAN1
locus, an 8-fold increase in the rate of chromosome V loss, and a 35-fold increase in the rate of mitotic recombination (Table ). Interestingly, each of these rates are considerably higher than those observed following the reduced expression of Pol1p (20
). One interpretation of this result is that the reduction in the level of DNA polymerase δ may reduce the efficiency of both replicative DNA synthesis (leading to elevated DSBs) and DNA synthesis associated with DNA repair, whereas reduced levels of DNA polymerase α may affect primarily replicative DNA synthesis (2
). The reduced expression of DNA polymerase δ also resulted in sensitivity to HU (Fig. ).
The mutator phenotype associated with low levels of DNA polymerase δ was also observed in our previous study (18
). Sequence analysis of the can1
mutations obtained in the strains with low levels of DNA polymerase δ indicated a somewhat different spectrum of mutations than that observed in the wild-type strain (18
). In the wild-type strain or the GAL
strain grown in high (0.05%) levels of galactose, most can1
mutations are single base changes, whereas in the GAL
strain grown in low (0.005%) levels of galactose, about half of the can1
mutations are small deletions with end points in short direct repeats (18
). Reduced levels of DNA polymerase δ may result in elevated levels of DNA polymerase slippage or an increased amount of synthesis by error-prone DNA polymerases. Since the spectrum of mutations in the strain with low levels of DNA polymerase and with mutations in rev3
does not differ significantly from the strain with low levels of DNA polymerase and wild-type REV3
, we argue in favor of the first possibility.
Reduced levels of DNA polymerase δ also substantially elevated (47-fold) illegitimate mating (Table ), although the degree of elevation is 4-fold less than that observed in strains with low levels of DNA polymerase α (20
). We previously proposed that reducing the expression of DNA polymerase α induces DSBs at fragile sites by stalling replication forks that then allow single-stranded DNA to form hairpin-like structures (20
); such structures would be readily formed in genomic regions such as FS2 that have inverted repeats (22
). A reduction in DNA polymerase δ might also be expected to have a destabilizing effect by uncoupling the replication of the lagging and leading strands. The observation that reduced levels of DNA polymerase α result in a stronger destabilization than that observed for DNA polymerase δ can be explained two ways. First, since DNA polymerase δ is a processive enzyme involved in the elongation stage of DNA replication (2
), whereas DNA polymerase α is the primase that is responsible for the synthesis of Okazaki fragments on the lagging strand template (2
), the cell may simply require more DNA polymerase α. Alternatively, DNA polymerase
may be able to partially substitute for reduced levels of DNA polymerase δ but not for reduced levels of DNA polymerase α. Finally, we note that the mechanism proposed for FS2-associated chromosome translocations in yeast is strikingly similar to that proposed for the frequently observed t(11,22) translocation in humans (6
Although there is a strong rationale for the mechanism by which FS2 functions as a fragile site, the mechanism by which FS1 acts is less clear. It is possible that DSBs initiated at FS2 are sometimes processed by nucleases, resulting in a recombinogenic structure ending in FS1. Consistent with this possibility, VanHulle et al. (28
) recently showed that a homothallism-induced DSB located 30 kb centromere distal to FS2 could stimulate chromosome rearrangements involving FS2. There are two other alternative possibilities. First, Ty elements, which are transcribed at high levels, may be prone to replication fork pausing, and this tendency could be stronger in conditions of perturbed DNA replication. Finally, it is possible that DSBs, other than the DSB at FS2, are random along the chromosome, but only those DSBs that occur in repetitive DNA elements result in chromosome rearrangements. DSBs in nonrepetitive DNA sequences could be repaired by sister chromatid recombination or by recombination with the homologous chromosome (class 3C events).
All of the chromosomal rearrangements observed in our analysis are consistent with the repair of a DSB in a Ty element of FS2 or FS1 by recombination with a Ty or delta element at an ectopic location (Table ). These results are similar to those that were previously reported in strains with reduced Pol1p expression (20
), and these observations argue that hindering DNA replication by different mechanisms can lead to the same type of chromosome rearrangements. Our results also reinforce the conclusions, from a variety of studies, that many of the chromosome rearrangements that occur in the lab and during the evolution of the yeast genome reflect Ty-Ty recombination (24
Chromosome fragile sites in human cells are hot spots for the chromosome rearrangements observed in cells derived from human cancers (3
). Although the DNA lesions responsible for fragile sites in mammalian cells have not yet been defined, one clear difference between mammalian and yeast fragile sites is the manner by which the initiating DNA lesion is repaired. In our studies, the translocations observed in strains with low DNA polymerase α or δ reflect homologous recombination between dispersed repeats. In contrast, the breakpoints of deletions associated with the mammalian fragile site FHIT
indicate that the recombinogenic lesions are repaired by nonhomologous end-joining (5
). Despite this difference, the identification of fragile sites in S. cerevisiae
may be useful in modeling some aspects of fragile sites in mammalian cells. For example, it would be interesting to determine whether the transient downregulation of DNA polymerase or a DNA replication cofactor could be responsible for some of the genetic instability associated with cancer cells.