Mutations in yeast replication proteins that increase CAG/CTG expansions also increase repeat fragility

Abstract

Expansion of trinucleotide repeats (TNRs) is the causative mutation in several human genetic diseases. Expanded TNR tracts are both unstable (changing in length) and fragile (displaying an increased propensity to break). We have investigated the relationship between fidelity of lagging-strand replication and both stability and fragility of TNRs. We devised a new yeast artificial chromomosme (YAC)-based assay for chromosome breakage to analyze fragility of CAG/CTG tracts in mutants deficient for proteins involved in lagging-strand replication: Fen1/Rad27, an endo/exonuclease involved in Okazaki fragment maturation, the nuclease/helicase Dna2, RNase HI, DNA ligase, polymerase delta, and primase. We found that deletion of RAD27 caused a large increase in breakage of short and long CAG/CTG tracts, and defects in DNA ligase and primase increased breakage of long tracts. We also found a correlation between mutations that increase CAG/CTG tract breakage and those that increase repeat expansion. These results suggest that processes that generate strand breaks, such as faulty Okazaki fragment processing or DNA repair, are an important source of TNR expansions.

FIG. 1.

The YAC breakage assay. Cells containing YAC CF1 (top; not to scale), which contains a CAG tract and URA3 gene, are FOA^S. If breakage occurs distal to the backup telomere (C₄A₄ tract)—for example, in the CAG tract (shown by a heavy line)—the YAC can be rescued by addition of a yeast telomere onto the C₄A₄ seed sequence. The resulting YAC has lost the URA3 gene and is thus FOA^R. The YAC is ∼62 kb long and contains 41 kb of lambda DNA between the LEU2 gene and C₄A₄ sequence.

FIG. 2.

Southern blot analysis of YACs from FOA^R colonies. Genomic DNA from FOA^S starting colonies (lanes 1 to 4) or FOA^R colonies from either wild-type (lanes 5 to 14) or rad27Δ (lanes 15 to 26) strains was purified, digested with BstEII, and separated on a 1% agarose gel. The YAC was visualized by hybridization to a probe to lambda DNA, which makes up the majority of the right arm of the YAC. BstEII restriction fragment sizes (in kilobases) predicted to hybridize to the probe are shown in a diagram at the bottom (not to scale) and to the left of the blot. The last BstEII fragment contains the backup telomere (C₄A₄), CAG tract, and URA3 gene, represented by boxes, and is predicted to be between 9 and 10 kb, depending on the size of the CAG tract (indicated by an arrow to the left of the gel). Breakage at the tip of the right arm—for example, at the CAG tract—and healing of the YAC by telomere addition at the backup telomere will reduce the size of the teminal band to approximately 6.6 kb (T). Healing at the CAG tract is expected to give a 7- to 7.5-kb terminal band (*). Healing behind the backup telomere sequence in the lambda sequence will result in loss of one or more of the bands, sometimes with appearance of new bands.

FIG. 3.

Breakage assay of YAC-CF1, containing CAG-0, -45, -70, or -155 tracts in different strain backgrounds. For each strain, 10 colonies of each tract length were grown separately for approximately three doublings at 30°C (which is semipermissive for the temperature-sensitive strains used) in Ura⁺ Leu⁻ medium to allow for breakage and healing at the backup telomere and then plated on FOA-Leu plates to select for breakage events and YC-Leu medium for a total cell count, and grown at the permissive temperature for each strain (23 or 25°C for temperature-sensitive mutants, 30°C for rnh35Δ and wild-type strains). Rates of FOA^R (10⁻⁶) were calculated by the method of the median (49). Rates that are significantly different from those of the wild type by a pooled variance t test are shown by asterisks (P < 0.05); standard errors are shown. (A) Wild-type strain. (B) Comparison of wild-type and rad27Δ strains. (C) Comparison of the wild-type strain and strains with mutations of other proteins involved in lagging-strand synthesis. Note differences in the scales for each graph.

FIG. 4.

Model for TNR expansions and breakage due to hairpin formation at flap structures. Displaced 5′ flaps, formed either on the lagging strand during replication or during nick or gap repair, that contain CTG repeats are predicted to fold into a hairpin structure at some frequency. The likelihood of hairpin formation is increased in strains defective for flap processing, such as strains lacking Fen1/Rad27. Four possible pathways for resolution of the hairpin/nick structure and their consequences for repeat instability are shown. NHEJ, nonhomologous end joining.