Sites of genetic instability in mitosis and cancer

Abstract

Certain chromosomal regions called common fragile sites are prone to difficulty during replication. Many tumors have been shown to contain alterations at fragile sites. Several models have been proposed to explain why these sites are unstable. Here we describe work to investigate models of fragile site instability using a yeast artificial chromosome carrying human DNA from a common fragile site region. In addition, we describe a yeast system to investigate whether repair of breaks at a naturally occurring fragile site in yeast, FS2, involves mitotic recombination between homologous chromosomes, leading to loss of heterozygosity (LOH). Our initial evidence is that repair of yeast fragile site breaks does lead to LOH, suggesting that human fragile site breaks may similarly contribute to LOH in cancer. This work is focused on gaining understanding that may enable us to predict and prevent the situations and environments that promote genetic changes that contribute to tumor progression.

Figure 1

Structure and characteristics of YAC 850a6 and its human DNA insert. (A) YAC 850a6 carries a 1.3Mb insert of DNA from human chromosome 3. The YAC also has a TRP1 marker gene and a yeast origin of replication (ARS) on the left arm, and URA3 and HPH markers on the right arm centromere-distal to the human DNA insert. (B) The human FHIT gene has ten exons, which are numbered and represented by short vertical bars. The FRA3B common fragile site is located within FHIT. The boundaries of this fragile site are not well defined, but there is general agreement that it is ~200 Kb in size and spans from FHIT intron 4 through part of intron 5. Red dotted lines indicate the portion of the FHIT/FRA3B region that are carried on YAC 850a6. (C) DNA flexibility in the FHIT gene from exon 3 through the portion of intron 5 carried on the YAC was analyzed using a 100bp sliding window. Regions with a twist angle deviation over 13.7° (top dotted line) are considered flexibility peaks because they are more than 4.5 standard deviations from the average flexibility. The locations of primer sets 1–16 used in the analysis of broken YACs are shown as yellow arrows. The locations of breaks in the YAC are identified by red squares. Each red square represents the last primer set to produce an amplified product from one of the 23 broken YACs analyzed.

Figure 2

System for analysis of mitotic recombination between homologous chromosomes. (A) The starting yeast strain is diploid and is homozygous for the ade2-1 mutation, which causes a block in the adenine biosynthesis pathway and accumulation of a red-pigmented intermediate molecule. Only yeast chromosome III from this diploid is shown. One copy of chromosome III contains the naturally occurring yeast fragile site FS2. The homologous copy of chromosome III does not contain this fragile site, but does have the SUP4-o tRNA gene inserted as shown. This tRNA suppresses the ade2-1 mutation, thus the starting strain is Ade⁺ and white in color. (B) A reciprocal crossover at the FS2 locus during mitotic cell division is shown. (C) In half of all reciprocal crossover events, chromosome segregation will result in the pattern shown, causing a red/white sectored colony in which chromosome III in each half of the sector is homozygous for all SNPs centromere-distal to the crossover. The left cell is red because the ade2-1 mutation is not suppressed (no SUP4-o gene is present). As a result of the crossover, both copies of the SUP4-o gene are in the right cell, making it white. Other mitotic recombination events that result in red/white sectoring, not shown but discussed in the text, include gene conversion and break-induced replication.