Destabilization of Yeast Micro- and Minisatellite DNA Sequences by Mutations Affecting a Nuclease Involved in Okazaki Fragment Processing (rad27) and DNA Polymerase δ (pol3-t)

Abstract

We examined the effects of mutations in the Saccharomyces cerevisiae RAD27 (encoding a nuclease involved in the processing of Okazaki fragments) and POL3 (encoding DNA polymerase ␦) genes on the stability of a minisatellite sequence (20-bp repeats) and microsatellites (1-to 8-bp repeat units). Both the rad27 and pol3-t mutations destabilized both classes of repeats, although the types of tract alterations observed in the two mutant strains were different. The tract alterations observed in rad27 strains were primarily additions, and those observed in pol3-t strains were primarily deletions. Measurements of the rates of repetitive tract alterations in strains with both rad27 and pol3-t indicated that the stimulation of microsatellite instability by rad27 was reduced by the effects of the pol3-t mutation. We also found that rad27 and pol3-01 (an allele carrying a mutation in the "proofreading" exonuclease domain of DNA polymerase ␦) mutations were synthetically lethal.All eukaryotic genomes thus far examined contain many simple repetitive DNA sequences, tracts of DNA with one or a small number of bases repeated multiple times (48). These repetitive regions can be classified as microsatellites (small repeat units in tandem arrays 10 to 60 bp in length) and minisatellites (larger repeat units in tandem arrays several hundred base pairs to several kilobase pairs in length). In this paper, arrays with repeat units 14 bp or less will be considered microsatellites and arrays with longer repeat units will be considered minisatellites.Previous studies show that simple repetitive sequences are unstable relative to "normal" DNA sequences, frequently undergoing additions or deletions of repeat units, in Escherichia coli (24), Saccharomyces cerevisiae (12), and mammals (59). This mutability has two important consequences. First, it results in polymorphic loci that are useful in genetic mapping and forensic studies (15,59). Second, although these repetitive tracts are usually located outside of coding sequences, alterations in the lengths of microsatellites or minisatellites located within coding sequences can produce frameshift mutations or novel protein variants (20,22,26).From studies of the effects of various mutations on microsatellite stability in yeast and E. coli (40) and the analysis of mutational changes caused by DNA polymerase in vitro (21), it is likely that most alterations reflect DNA polymerase slippage events (47). These events involve the transient dissociation of the primer and template strands during the replication of a microsatellite (Fig. 1). If the strands reassociate to yield an unpaired repeat on the primer strand, the net result is an addition of repeats (following a second round of DNA replication). Unpaired repeats on the template strand would result in a deletion by the same mechanism.A number of mutations have been shown to elevate microsatellite instability. In E. coli (24, 46), yeast (44, 45), and mammalian cells (27), mutations in genes affecting DNA mismatch repair dramatically elevate the ins...

Meiotic instability of CAG repeat tracts occurs by double-strand break repair in yeast

“…During DNA replication, the hairpin formation on the template strand or on the newly synthesized strand would produce a deletion or an expansion of the repeat tract, respectively. This hypothesis is consistent with the observation that CAG-repeat instability depends on the direction in which the replication fork proceeds through the repeat tract (8,9,13,20).The repeat tract instability is also higher in the rad27-deletion mutant, which fails to process Okazaki fragments (12,14,21,22). A failure to process the Okazaki fragments may facilitate hairpin formation on the newly synthesized strand.…”

“…Several studies using model organisms indicate that replication slippage plays a major role in the repeat tract instability (7)(8)(9)(10)(11)(12)(13)(14). The latter model is also supported by the in vitro observations that CAG repeats can form hairpin structures, with the CTG hairpin being more stable than the CAG hairpin (15)(16)(17)(18).…”

“…The repeat tract instability is also higher in the rad27-deletion mutant, which fails to process Okazaki fragments (12,14,21,22). A failure to process the Okazaki fragments may facilitate hairpin formation on the newly synthesized strand.…”

Concerted Action of Exonuclease and Gap-dependent Endonuclease Activities of FEN-1 Contributes to the Resolution of Triplet Repeat Sequences (CTG) - and (GAA) -derived Secondary Structures Formed during Maturation of Okazaki Fragments

“…This suggests that secondary structures can form in vivo and impair replication/repair enzymes. Studies in model organisms, such as S. cerevisiae, have shown that mutations in DNA replication enzymes, particularly Rad27 (yeast FEN-1 homolog), lead to TNR instability (6,(22)(23)(24)(25)(26)(27)(28). Rad27/FEN-1 is a multifunctional nuclease that plays a critical role in maintaining genome stability through RNA primer removal and long patch base excision repair.…”

Saccharomyces cerevisiae RAD27 complements its Escherichia coli homolog in damage repair but not mutation avoidance

“…In assays that measure chromosome instability, rad27 deletion strains are associated with di-and trinucleotide repeat instability (Johnson et al 1995), and a strong mutator phenotype (Tishkoff et al 1997;Johnson et al 1995;Kokoska et al 1998). The mutator phenotype is characterized by a 50-fold increase in the CAN1 gene mutation rate, with a bias toward duplication mutations.…”