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. 2004 Jun 15;101(24):9039-44.
doi: 10.1073/pnas.0403093101. Epub 2004 Jun 7.

Mutator genes for suppression of gross chromosomal rearrangements identified by a genome-wide screening in Saccharomyces cerevisiae

Stephanie Smith  1 , Ji-Young Hwang, Soma Banerjee, Anju Majeed, Amitabha Gupta, Kyungjaem Myung
Affiliations

Mutator genes for suppression of gross chromosomal rearrangements identified by a genome-wide screening in Saccharomyces cerevisiae

Stephanie Smith et al. Proc Natl Acad Sci U S A. .

Abstract

Different types of gross chromosomal rearrangements (GCRs), including translocations, interstitial deletions, terminal deletions with de novo telomere additions, and chromosome fusions, are observed in many cancers. Multiple pathways, such as S-phase checkpoints, DNA replication, recombination, chromatin remodeling, and telomere maintenance that suppress GCRs have been identified. To experimentally expand our knowledge of other pathway(s) that suppress GCRs, we developed a generally applicable genome-wide screening method. In this screen, we identified 10 genes (ALO1, CDC50, CSM2, ELG1, ESC1, MMS4, RAD5, RAD18, TSA1, and UFO1) that encode proteins functioning in the suppression of GCRs. Moreover, the breakpoint junctions of GCRs from these GCR mutator mutants were determined with modified breakpoint-mapping methods. We also identified nine genes (AKR1, BFR1, HTZ1, IES6, NPL6, RPL13B, RPL27A, RPL35A, and SHU2) whose mutations generated growth defects with the pif1Delta mutation. In addition, we found that some of these mutations changed the telomere size.

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Figures

Fig. 1.
Fig. 1.
Genome-wide screening to find GCR mutators. (A) A library composed of strains carrying the GCR assay system, a pif1Δ mutation, and 1 of 4,644 gene deletion mutations was generated. (B) Secondary screening identified three types of GCR mutator mutations (see Table 1). (C) Screen results for CAN and GCR (CAN-5FOA) mutator screens. Primary screen is shown in Left, and secondary screen is shown in Right. In the CAN mutator assay, only one plate was used for the primary screen. Putative GCR mutators were selected only when two independent FC plates generated more than five colonies.
Fig. 2.
Fig. 2.
Rearranged breakpoint junctions were determined with three PCR steps. (A) Genome walking PCR was performed to narrow the breakpoint junctions to within 400 bp. WT shows PCR products generated from an intact chromosome V. PCR patterns generated with three different chromosomes isolated from mutants carrying GCRs were visualized in 2% agarose gel. (B) De novo telomere-addition PCR identified GCRs carrying a terminal deletion, followed by telomere sequence addition. Chromosomal DNAs from wild type (WT) and Mt, representing intact chromosome V and clones carrying GCRs, respectively, were used as templates for PCR. (C) The linker-mediated PCR was performed with chromosomal DNA that did not generate ladder bands in the de novo telomere-addition PCR. Chromosomal DNAs from WT and GCR clone (Mt) were digested with different restriction enzyme (RE) and ligated with linkers. Then, a primer that can bind at the end of remaining chromosome V of GCR clone and a linker-specific primer were used for amplification of rearranged chromosomes.
Fig. 3.
Fig. 3.
There are at least five different steps for GCR suppression and formation. Although genomes are highly protected, spontaneous DNA damages due to cellular errors can be generated. Inactivation of proteins such as ALO1, ELG1, TSA1, MMS4, and MUS81 may cause an increase in spontaneous DNA damage. The DNA damage that is generated is detected first by the DNA damage-sensing machinery. The S-phase checkpoints sense this damage and transfer the signal to the DNA repair machinery to facilitate repair. RAD5, RAD18, MUS81, and MMS4 seem to function in DNA repair as well as in the sensing of DNA damage. RAD5 and RAD18, along with UFO1, might function in the transfer of signal by means of the ubiquitination of substrates. There are many different DNA repair pathways that are used to fix broken DNA. Break-induced replication, postreplication repair, and MUS81/MMS4-mediated recombination repair seem to play major roles in the suppression of GCRs. DNA damage that is not corrected by DNA repair pathways is sensed by the mitotic checkpoint, and the GCR formation machinery becomes engaged. First, damaged DNA is processed by unknown endonucleases or exonucleases to generate appropriate substrates. Then, telomerase adds telomere sequences at the end of broken chromosomes to produce the de novo telomere addition that is inhibited by PIF1 or translocations with other chromosome sequences, or they are generated by LIG4-dependent or -independent pathways.
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