Implementation of an extrachromosomal system for the detection of novel DNA double strand break repair genes in S. cerevisiae

Caputo Galarce, Valentina (2003). Implementation of an extrachromosomal system for the detection of novel DNA double strand break repair genes in S. cerevisiae. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000f563

Abstract

The outcome of DNA damage is diverse and generally adverse. Acute effects arise from disturbed DNA metabolism, triggering cell-cycle arrest or cell death. Long-term effects result from irreversible mutation contributing to oncogenesis and genome instability. In view of the many types of lesions, several pathways were developed to repair these lesions: nucleotide-excision repair (NER), base-excision repair (BER), mismatch repair (MMR), homologous recombination (HR) and non-homologous end joining (NHEJ). The double-strand break (DSB) is the most dangerous type of DNA lesion, it can be repaired mainly by HR (by crossover (CO), gene conversion (GC), and singlestrand annealing (SSA)) or by non-homologous end-joining (NHEJ). When, after replication a second identical DNA copy is available, HR seems to be preferred, other wise cells rely on NHEJ, which is more error prone. Up to now, all DSB repair processes have been studied separately, although it is clear that all of them can occur simultaneously in different proportions. We have created a new molecular plasmid system to simultaneously detect all four types of recombinational DBS repair. To this end, we constructed an in vivo/in vitro HNS plasmid system (HNS: HR, NHEJ, SSA), based upon two topologically different DNA molecules, which allows us to follow all four recombination processes at once. The plasmids, named pURRA8A and pRURA8A, contain two truncated non-functional URA3 genes in direct or inverted orientation respectively, sharing a central homologous region where a l-Scel site was introduced artificially. The plasmids also carry a centromere sequence and two phenotypic markers TRP1 and ADE8. DSB can be induced in vitro at the homologous (l-SceI) or non-homologous (BamHI) region. Yeast transformation with linearized plasmids was performed in strain YPH 250 and isogenic knockout strains lacking either the RAD52 gene involved in HR and SSA, HDF1 (yKU70) and NEJ1 involved in NHEJ. In addition MRE11 complex and MSH2 null mutants were studied. Distribution of DSB repair events among the various pathways was monitored by phenotypic and PCR analysis. The rad52 knockout mutant showed lower levels of CO and SSA, while the hdf1 mutant showed a decrease in conservative and non conservative NHEJ, as expected. These results confirm the validity of the HNS system for monitoring all 4 repair pathways simultaneously. The HNS system has been used to identify new genes involved in DNA damage response. DBS were induced in vivo by the expression of the l-Sce I endonuclease under Gal1-promoter control. After transformation, we performed transposon mutagenesis using the mTn-lacZ/LEU2 library system and selected cells that lost the ability to recombine. From the initial 7000 mutants tested, we selected initially 150 that were rescreening to obtain finally 33 mutants. The identification of the locus of the transposon insertion of all of them was performed. Some known genes (RAD50, SWR1, MCK1, SIN4, RSC2, SWE1 and DBP1) as well as unknown ones (YLR238W, YLR089C, YMR278W) were selected. Null mutants of all them were constructed, DSBR events profile as well as MMS sensitivity were determined. Relative rates of DSB repaired by HR, NHEJ and SSA were examined in all the selected null mutants strains. By comparing the distribution of DSBR by different mechanisms, we were able to obtain a strain-specific profile in which the relative proportions of repair events occurring in the cell were characteristic of that mutation. RSC2 (RSC complex component) and YLR235w (FHA containing protein) genes were selected to further characterisation. Epistatic null mutants analysis with key recombination genes (yku70 and rad52) was performed. MMS sensitivity and survival was analysed, as well as DSB induction in-vivo. This enables us to verify the involvement of a particular gene product in DNA damage response. In particular, we showed that Ylr238w is involved in DNA damage transcription regulation.

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