Ionizing radiation and restriction enzymes induce microhomology-mediated illegitimate recombination in Saccharomyces cerevisiae

Abstract

DNA double-strand breaks can be repaired by illegitimate recombination without extended sequence homology. A distinct mechanism namely microhomology-mediated recombination occurs between a few basepairs of homology that is associated with deletions. Ionizing radiation and restriction enzymes have been shown to increase the frequency of nonhomologous integration in yeast. However, the mechanism of such enhanced recombination events is not known. Here, we report that both ionizing radiation and restriction enzymes increase the frequency of microhomology-mediated integration. Irradiated yeast cells displayed 77% microhomology-mediated integration, compared to 27% in unirradiated cells. Radiation-induced integration exhibited lack of deletions at genomic insertion sites, implying that such events are likely to occur at undamaged sites. Restriction enzymes also enhanced integration events at random non-restriction sites via microhomology-mediated recombination. Furthermore, generation of a site-specific I-SceI-mediated double-strand break induces microhomology-mediated integration randomly throughout the genome. Taken together, these results suggest that double-strand breaks induce a genome-wide microhomology-mediated illegitimate recombination pathway that facilitates integration probably in trans at non-targeted sites and might be involved in generation of large deletions and other genomic rearrangements.

Figure 1.

Target sequences of NHI events of BgIII-linearized pM151 in wild-type strain RSY12. (A) Spontaneous events. (B) After exposure to 50Gy of γ-irradiation. Shown are the plasmid DNA substrate, the target sequences and the genomic locations into which the plasmid integrated. The ends of the integrating DNA are shown above and below the genomic target sequences. The numbers 1 and 2 correspond to the sequence of the 5′ (1) and 3′ (2) PSS ends of the integrating DNA. The region of microhomology between the ends of integrating DNA and genomic target DNA is visualized by vertical dashed lines, with the crossover occurring at either one of the dashed lines. If there is no overlapping microhomology sequence between the recombining DNAs, the site of crossover is represented by vertical solid line. End 1 of the integrating DNA sequence is shown as single underlined sequence and after the crossover continuing at the target. End 2 is double underlined continuing after the crossover. The genomic location, locus and gene of the target sites are as follows: C8, Chr. (Chromosome) I, promoter of YAL022C, FUN26; C9, Chr. VI, promoter of tA(AGC), tRNA; C15, Chr. XVI, YPL070W, MUK1; C17, CHR. VIII, YIL071C, PCI8; C19, Chr. XII, RDN37-1, 35SrRNA; C20, Chr. XII, RDN5-1, 5SrRNA; C22, Chr. II, promoter of YBL079W, NUP170; C23, Chr. VI, hypo. (hypothetical) ORF; C24, Chr. XVI, YPR124W, CTR1; C28, Chr. XI 151654, intergenic region; C30, Chr. XIV, YNL012W, SPO1; C36, Chr. XVI 630665, intergenic region; C37, Chr. XIV 669282, intergenic region; R2, Chr. IV, YDR131C, hypo. ORF; R3, Chr. XIII, promoter of YMR251W, GTO3; Chr. XV, ROL089C, HAL9; R7, Chr. V, YER135C hypo. ORF/Chr. XII, RDN5-1, 5SrRNA; R9, Chr. XV, YOR313C, SPS4/Chr. XIII, YMR310C, hypo. ORF; R10, Chr. II, promoter of YBR001C, NTH2; R11, Chr. V, YER135C, hypo. ORF; R12, Chr. IV, YKR017C, hypo. ORF; R21, Chr. II, YBL060W, hypo. ORF; R22, Chr. II, YBL060W, hypo. ORF; R23, Chr. IV, YDR258C, HSP78.

Figure 2.

The distribution of microhomology usage in spontaneous and radiation-induced NHI events in strain RSY12.

Figure 3.

Target sequences of restriction enzyme-induced NHI events. In Asp718-KpnI (AK) events, KpnI was added to Asp718-linearized plasmid pM150 during transformation. In EcoRI-BglII (RB) events, BglII was added to EcoRI-linearized plasmid pM150. In Asp718 (filled)-KpnI (AFK) events, the ends of Asp718-linearized plasmid pM150 were filled before transformation. KpnI was added to the transformation mixture. The genomic location, locus and gene of the target sites are as follows: AK1, Chr. VII, YGL086W, MAD1; AK3, Intergenic region; AK8, Chr. XII, YLR207W, HRD3; AK2, intergenic region; AK13, Chr. XV, YOR269W, PAC1; AK15, Chr. II, YBR176W, ECM31; AK16, YDR205C; AK20, Chr. VII, YGL081W, hypo. ORF; RB11, intergenic region; RB12, Chr. X, YJR036C, HUL4; AFK11, Chr. IX, YIL085C, KTR7; AFK12, Chr. IV, YDL197C, ASF2.

Figure 4.

Target sites of NHI events of KpnI-linearized pM150 (3′PSS) in wild-type strain RSY12. (A) Spontaneous events. (B) After co-transformation with BglII. The genomic location, locus and gene of the target sites are as follows: KC15, Chr. III, YCL067C, HMLALPHA2; KC16, Chr. IX, YIL137C, RBF108; KC17, Chr. XI, YKL197C, PEX1; KC18, Chr. II, 21590, intergenic region; KC19, Chr. XIV, YNL298W, CLA4; KC20, Chr. II, YBL104C, hypo. ORF; KC21, Chr. VII, 144125, intergenic region; KB2, Chr. VII, YGL062W, PYC1; KB8, Chr. IV, YDL042C, SIR2; KB9, Chr. III, YCR061W, hypo. ORF; KB11, Chr. IV, 11262, intergenic region; KB10, Chr. XIII, YML043C, RRN11; KB71, Chr. III, YCL057W, PRD1; KB75, Chr. XIII, YML086C, ALO1.

Figure 5.

Target sites of NHI events of BglII-linearized pM151 in (A) YWY200 strain in the absence of the I-SceI site in galactose-growing medium. (B) YWY200-SceI strain in the presence of the I-SceI site at Chr. IV upon galactose induction of I-SceI endonuclease. The genomic location, locus and gene of the target sites are as follows: Y8, Chr. V, ARS521; Y12, Chr. XII, YLR306W, UBC12; Y15, Chr. XI, YKL044W, hypo. ORF; Y24, Chr. VII, YGR148C, RPL24B; Y26, Chr. XII, NTS1-2; G11, Chr. XII, NTS1-2; G13, Chr. VII, YGR142W, BTN2; G14, Chr. XII, RDN37-2, a 939 bp deletion; G21, Chr. IV, YDR030C, RAD28; G23, Chr. VI, YFL065C, hypo, ORF; G25, Chr. XII, RDN25-2; G26, Chr. XIII, YML103C, NUP188; G27, Chr. VII, YGL131C, SNT2; G38, Chr. XVIII, YML103C, NUP188; G39, Chr. II, YBR140C, IRA1.

Figure 6.

The distribution of microhomology usage in I-SceI-induced NHI events in the absence and presence of the I-SceI site.