Genome wide distribution of illegitimate recombination events in Kluyveromyces lactis.
Illegitimate recombination (IR) is the process by which two DNA molecules not sharing homology to each other are joined. In Kluyveromyces lactis, integration of heterologous DNA occurred very frequently therefore constituting an excellent model organism to study IR. IR was completely dependent on the nonhomologous end-joining (NHEJ) pathway for DNA double strand break (DSB) repair and we detected no other pathways capable of mediating IR. NHEJ was very versatile, capable of repairing both blunt and non-complementary ends efficiently. Mapping the locations of genomic IR-events revealed target site preferences, in which intergenic regions (IGRs) and ribosomal DNA were overrepresented six-fold compared to open reading frames (ORFs). The IGR-events occurred predominantly within transcriptional regulatory regions. In a rad52 mutant strain IR still preferentially occurred at IGRs, indicating that DSBs in ORFs were not primarily repaired by homologous recombination (HR). Introduction of ectopic DSBs resulted in the efficient targeting of IR to these sites, strongly suggesting that IR occurred at spontaneous mitotic DSBs. The targeting efficiency was equal when ectopic breaks were introduced in an ORF or an IGR. We propose that spontaneous DSBs arise more frequently in transcriptional regulatory regions and in rDNA and such DSBs can be mapped by analyzing IR target sites.
Department of Developmental Biology, Wennergren Institute, Stockholm University, SE-106 91 Stockholm, Sweden.
Andreas
A
Kegel
Paula
P
Martinez
Sidney D
SD
Carter
Stefan U
SU
Aström
D002869:Chromosome Aberrations; D004249:DNA Damage; D004260:DNA Repair; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D016681:Genome, Fungal; D007716:Kluyveromyces / Q000235:genetics*; D009154:Mutation; D011003:Ploidies; D051721:Rad52 DNA Repair and Recombination Protein / Q000235:genetics; D011995:Recombination, Genetic; D050436:Regulatory Elements, Transcriptional; D014158:Transcription, Genetic
D051721:Rad52 DNA Repair and Recombination Protein; D004264:DNA Topoisomerases, Type I