Replication Slippage between Distant Short Repeats in Saccharomyces cerevisiae Depends on the Direction of Replication and the RAD50 and RAD52 Genes

Abstract

Small direct repeats, which are frequent in all genomes, are a potential source of genome instability. To study the occurrence and genetic control of repeat-associated deletions, we developed a system in the yeast Saccharomyces cerevisiae that was based on small direct repeats separated by either random sequences or inverted repeats. Deletions were examined in the LYS2 gene, using a set of 31- to 156-bp inserts that included inserts with no apparent potential for secondary structure as well as two quasipalindromes. All inserts were flanked by 6- to 9-bp direct repeats of LYS2 sequence, providing an opportunity for Lys+ reversion via precise excision. Reversions could arise by extended deletions involving either direct repeats or random sequences and by -1-or +2-bp frameshift mutations. The deletion breakpoints were always associated with short (3- to 9-bp) perfect or imperfect direct repeats. Compared with the POL+ strain, deletions between small direct repeats were increased as much as 100-fold, and the spectrum was changed in a temperature-sensitive DNA polymerase delta pol3-t mutant, suggesting a role for replication. The type of deletion depended on orientation relative to the origin of replication. On the basis of these results, we propose (i) that extended deletions between small repeats arise by replication slippage and (ii) that the deletions occur primarily in either the leading or lagging strand. The RAD50 and RAD52 genes, which are required for the recombinational repair of many kinds of DNA double-strand breaks, appeared to be required also for the production of up to 90% of the deletions arising between separated repeats in the pol3-t mutant, suggesting a newly identified role for these genes in genome stability and possibly replication.

Distinct Function of Conserved Amino Acids in the Fingers of Saccharomyces cerevisiae DNA Polymerase α

“…This result is consistent with other recent reports showing genetic instability in cells with mutations in DNA polymerases or replication-associated proteins (51)(52)(53)). An increased frequency of direct repeat-associated deletions was also reported with a temperature-sensitive pol ␣ allele in Schizosaccharomyces pombe (52).…”

The sbcC and sbcD genes of Escherichia coli encode a nuclease involved in palindrome inviability and genetic recombination

“…However, studies of DNA replication and double-strand break formation during meiosis reveal a significant temporal overlap between these reactions (Padmore et al 1991) suggesting that a mechanism exists to ensure that cleavage occurs after replication. Whether DNA secondary structure plays any part in the generation of double-strand breaks in yeast is unknown but rad50 mutants reverse the instability of inverted repeats observed in a pol3 mutant (Gordenin et al 1992;Tran et al 1995). These results also suggest a role for RAD50 in DNA replication (Tran et al 1995).…”

Transcription-associated mutagenesis in yeast is directly proportional to the level of gene expression and influenced by the direction of DNA replication

“…Pol32 is a subunit of DNA Polδ and the deletion of the POL32 gene results in reduced Polδ processivity and a compromised DNA damage response [39]. Similarly, in a strain with mutation in the major catalytic subunit of the Polδ (pol3-t mutant), a large orientationindependent increase in the deletion frequency of sequences flanked by 6-7 bp direct repeats has been reported [40]. It has been suggested that large dels reflect the inherent single-stranded character of the lagging-strand template, which would be expected to be further enhanced if polymerization is compromised, allowing the formation of misaligned slippage intermediates between nontandem direct repeats [41].…”