Academic journal article Genetics

Genetic Instability Induced by Overexpression of DNA Ligase I in Budding Yeast

Academic journal article Genetics

Genetic Instability Induced by Overexpression of DNA Ligase I in Budding Yeast

Article excerpt

ABSTRACT

Recombination and microsatellite mutation in humans contribute to disorders including cancer and trinucleotide repeat (TNR) disease. TNR expansions in wild-type yeast may arise by flap ligation during lagging-strand replication. Here we show that overexpression of DNA ligase I (CDC9) increases the rates of TNR expansion, of TNR contraction, and of mitotic recombination. Surprisingly, this effect is observed with catalytically inactive forms of Cdc9p protein, but only if they possess a functional PCNA-binding site. Furthermore, in vitro analysis indicates that the interaction of PCNA with Cdc9p and Rad27p (Fen1) is mutually exclusive. Together our genetic and biochemical analysis suggests that, although DNA ligase I seals DNA nicks during replication, repair, and recombination, higher than normal levels can yield genetic instability by disrupting the normal interplay of PCNA with other proteins such as Fen1.

EXPANSION of trinucleotide repeat (TNR) tracts can cause a significant number of hereditary neurological disorders such as Huntington disease (HD), myotonic dystrophy, and fragile X syndrome (reviewed in CLEARY and PEARSON 2003; LENZMEIER and FREUDENREICH 2003). Studies in budding yeast have supported a number of different models for the expansion and contraction of TNR tracts including classical "slippage" and recombination (MAURER et al. 1996, 1998; FREUDENREICH et al. 1997, 1998; MIRET et al. 1997, 1998; COHEN et al. 1999; RICHARD et al. 1999, 2000, 2003; SCHWEITZER and LIVINGSTON 1999; IRELAND et al. 2000; JANKOWSKI et al. 2000; SCHWEITZER et al. 2001; JANKOWSKI and NAG 2002; CALLAHAN et al. 2003; CLEARY and PEARSON 2003; LENZMEIER and FREUDENREICH 2003). In all models, the ability of TNR sequences to produce intermediates stabilized by secondary structure formation is thought to be critical to the mutation process (reviewed in MITAS 1997; CLEARY and PEARSON 2003; LENZMEIER and FREUDENREICH 2003).

Studies with yeast strains lacking the gene coding for flap endonuclease Fenl (rad27A strains) show enhanced levels of genetic instability that are thought to result from inefficient processing of single-strand DNA flaps formed during lagging-strand DNA replication (TiSHKOFF et al. 1997). During Okazaki fragment synthesis, the 5'-end of a downstream Okazaki fragment may be made single stranded by strand displacement synthesis as Pol ä extends the adjacent upstream Okazaki fragment. The 5'-flap thus formed would normally be eliminated by a combination of enzymes including Fenl (BuDD et al 1995; BAE and SEO 2000; BAE et al. 2001; MAGA et al. 2001; AYYAGARI et al. 2003). The absence of Fenl could yield DNA duplications by slipped mispairing of the flap followed by repair or replication prior to repair (TISHKOFF et al. 1997). Unprocessed flaps could also cause an increase in DNA breaks (SYMINGTON 1998) and lead to greater genetic instability (TISHKOFF et al. 1997).

This model has been extended to TNR mutations in wild-type cells (GORDENIN et al. 1997). Expansion mutations were suggested to arise when a newly formed and an as-yet unprocessed TNR-containing flap forms a secondary structure that makes it possible for its 5'-end to directly ligate to the 3'-end of the upstream Okazaki fragment. Support for this model comes from data showing that single-stranded flaps containing a TNR secondary structure [e.g., (GTG)n] are resistant to the action of Fenl in vitro (SpiRO et al. 1999; HENRICKSEN et al. 2000). A delay in flap processing in vivo could increase the chance that the unprocessed flap could be ligated back into the nascent strand resulting in an expansion mutation (GORDENIN et al. 1997). In fact, delayed flap processing in rad27A cells results in enhanced rates of TNR expansion and contraction mutations (FREUDENREICH et al. 1998; SCHWEITZER and LIVINGSTON 1998, 1999; SPIRO et al. 1999; IRELAND et al. 2000; ROLFSMEIER et al. 2000, 2001; CALLAHAN et al. 2003; LIU et al. 2004). An unprocessed flap could also give rise to a double-strand break (DSB) that, during recombinational repair, might lead to expansions or contractions (GoRDENiN et al. …

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