Effects of mutations in SGS1 and in genes functionally related to SGS1 on inverted repeat-stimulated spontaneous unequal sister-chromatid exchange in yeast

Abstract

Background: The presence of inverted repeats (IRs) in DNA poses an obstacle to the normal progression of the DNA replication machinery, because these sequences can form secondary structures ahead of the replication fork. A failure to process and to restart the stalled replication machinery can lead to the loss of genome integrity. Consistently, IRs have been found to be associated with a high level of genome rearrangements, including deletions, translocations, inversions, and a high rate of sister-chromatid exchange (SCE). The RecQ helicase Sgs1, in Saccharomyces cerevisiae, is believed to act on stalled replication forks. To determine the role of Sgs1 when the replication machinery stalls at the secondary structure, we measured the rates of IR-associated and non-IR-associated spontaneous unequal SCE events in the sgs1 mutant, and in strains bearing mutations in genes that are functionally related to SGS1.

Results: The rate of SCE in sgs1 cells for both IR and non-IR-containing substrates was higher than the rate in the wild-type background. The srs2 and mus81 mutations had modest effects, compared to sgs1. The exo1 mutation increased SCE rates for both substrates. The sgs1 exo1 double mutant exhibited synergistic effects on spontaneous SCE. The IR-associated SCE events in sgs1 cells were partially MSH2-dependent.

Conclusions: These results suggest that Sgs1 suppresses spontaneous unequal SCE, and SGS1 and EXO1 regulate spontaneous SCE by independent mechanisms. The mismatch repair proteins, in contradistinction to their roles in mutation avoidance, promote secondary structure-associated genetic instability.

Figure 1

Unequal SCE assay. A. The his3 substrate for measurement of unequal SCE. The his3-Δ3' construct is marked with a tail, and the his3-Δ5' construct is marked with an arrowhead. The shaded region indicates the regions shared by the two deletion constructs. Expanded region under the linear map represents the palindromic insertion. B. DSB repair by gene conversion. A DSB is formed when the replication fork has stalled at the secondary structure. Although a secondary structure can form on both the lagging and the leading strand, the discontinuous nature of DNA synthesis is likely to facilitate formation of greater amounts of secondary structures on the lagging strand than on the leading strand. Shown here is the repair of a DSB formed on the lagging strand via unequal SCE, using the sister chromatid as a template. The unequal SCE events generate a wild-type HIS3 gene. DSBs can also be repaired by equal SCE. However, equal SCE will not give rise to a wild-type HIS3 gene. DSBs are likely to form via the endonuclease activity of a structure-specific nuclease.