Cotranscriptionally formed DNA:RNA hybrids mediate transcription elongation impairment and transcription-associated recombination.

Genetic instability, a phenomenon relevant for developmentally regulated processes, cancer, and inherited disorders, can be induced by transcription. However, the mechanisms of transcription-associated genetic instability are not yet understood. Analysis of S. cerevisiae mutants of THO/TREX, a conserved eukaryotic protein complex functioning at the interface of transcription and mRNA metabolism, has provided evidence that transcription elongation impairment can cause hyperrecombination. Here we show, using hpr1Delta mutants, that the nascent mRNA can diminish transcription elongation efficiency and promote recombination. If during transcription the nascent mRNA is self-cleaved by a hammerhead ribozyme, the transcription-defect and hyperrecombination phenotypes of hpr1Delta cells are suppressed. Abolishment of hyperrecombination by overexpression of RNase H1 and molecular detection of DNA:RNA hybrids indicate that these are formed cotranscriptionally in hpr1Delta cells. These data support a model to explain the connection between recombination, transcription, and mRNA metabolism and provide a new perspective to understanding transcription-associated recombination.

[1]  Kevin Struhl,et al.  TREX is a conserved complex coupling transcription with messenger RNA export , 2002, Nature.

[2]  M. Raymond,et al.  RNase H Overproduction Corrects a Defect at the Level of Transcription Elongation during rRNA Synthesis in the Absence of DNA Topoisomerase I in Escherichia coli * , 2000, The Journal of Biological Chemistry.

[3]  M. Rosbash,et al.  Interactions between mRNA Export Commitment, 3′-End Quality Control, and Nuclear Degradation , 2002, Molecular and Cellular Biology.

[4]  A. Aguilera,et al.  Differential intrachromosomal hyper-recombination phenotype of spt4 and spt6 mutants of S. cerevisiae , 1996, Current Genetics.

[5]  R. Müller,et al.  Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. , 1995, Gene.

[6]  U. Storb,et al.  Somatic hypermutation of immunoglobulin genes is linked to transcription initiation. , 1996, Immunity.

[7]  Takashi Horiuchi,et al.  Transcription-dependent recombination and the role of fork collision in yeast rDNA. , 2003, Genes & development.

[8]  H. Fan,et al.  High-Copy-Number Expression of Sub2p, a Member of the RNA Helicase Superfamily, Suppresses hpr1-Mediated Genomic Instability , 2001, Molecular and Cellular Biology.

[9]  M. Itaya,et al.  The absence of ribonuclease H1 or H2 alters the sensitivity of Saccharomyces cerevisiae to hydroxyurea, caffeine and ethyl methanesulphonate: implications for roles of RNases H in DNA replication and repair , 2000, Genes to cells : devoted to molecular & cellular mechanisms.

[10]  Rodney Rothstein,et al.  Elevated recombination rates in transcriptionally active DNA , 1989, Cell.

[11]  R. Cedergren,et al.  A small nucleolar RNA:ribozyme hybrid cleaves a nucleolar RNA target in vivo with near-perfect efficiency. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[12]  A. Furger,et al.  Integrating mRNA Processing with Transcription , 2002, Cell.

[13]  Trevor Lithgow,et al.  A protein complex containing Tho2, Hpr1, Mft1 and a novel protein, Thp2, connects transcription elongation with mitotic recombination in Saccharomyces cerevisiae , 2000, The EMBO journal.

[14]  R. Moses.  DNA damage processing defects and disease. , 2001, Annual review of genomics and human genetics.

[15]  Paul Tempst,et al.  Nab2p and the Thp1p-Sac3p Complex Functionally Interact at the Interface between Transcription and mRNA Metabolism* , 2003, Journal of Biological Chemistry.

[16]  R. G. Lloyd,et al.  Modulation of RNA Polymerase by (p)ppGpp Reveals a RecG-Dependent Mechanism for Replication Fork Progression , 2000, Cell.

[17]  Andrés Aguilera,et al.  A novel yeast gene, THO2, is involved in RNA pol II transcription and provides new evidence for transcriptional elongation‐associated recombination , 1998, The EMBO journal.

[18]  R. Kolodner,et al.  Saccharomyces cerevisiae chromatin-assembly factors that act during DNA replication function in the maintenance of genome stability , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[19]  A. S. Krasilnikov,et al.  Transcription through a simple DNA repeat blocks replication elongation , 1998, The EMBO journal.

[20]  R. Luna,et al.  The yeast THO complex and mRNA export factors link RNA metabolism with transcription and genome instability , 2002, The EMBO journal.

[21]  A. Aguilera,et al.  Recombinogenic effects of DNA-damaging agents are synergistically increased by transcription in Saccharomyces cerevisiae. New insights into transcription-associated recombination. , 2003, Genetics.

[22]  R. Crouch,et al.  RNase H1 of Saccharomyces cerevisiae: methods and nomenclature. , 2001, Methods in enzymology.

[23]  M. Lieber,et al.  R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells , 2003, Nature Immunology.

[24]  S. Chávez,et al.  The yeast HPR1 gene has a functional role in transcriptional elongation that uncovers a novel source of genome instability. , 1997, Genes & development.

[25]  F. Alt,et al.  Transcription-induced Cleavage of Immunoglobulin Switch Regions by Nucleotide Excision Repair Nucleases in Vitro* , 2000, The Journal of Biological Chemistry.

[26]  J. Sogo,et al.  Rad52-Independent Accumulation of Joint Circular Minichromosomes during S Phase in Saccharomyces cerevisiae , 2003, Molecular and Cellular Biology.

[27]  A. Aguilera.  The connection between transcription and genomic instability , 2002, The EMBO journal.

[28]  K. Khanna,et al.  DNA double-strand breaks: signaling, repair and the cancer connection , 2001, Nature Genetics.

[29]  S. Ehrlich,et al.  RuvAB Acts at Arrested Replication Forks , 1998, Cell.

[30]  É. Massé,et al.  Escherichia coli DNA Topoisomerase I Inhibits R-loop Formation by Relaxing Transcription-induced Negative Supercoiling* , 1999, The Journal of Biological Chemistry.

[31]  T. Ushiki,et al.  Molecular Visualization of Immunoglobulin Switch Region RNA/DNA Complex by Atomic Force Microscope* , 2003, The Journal of Biological Chemistry.

[32]  T. Honjo,et al.  Switch region of immunoglobulin Cμ gene is composed of simple tandem repetitive sequences , 1981, Nature.

[33]  C. Peterson,et al.  HPR1 encodes a global positive regulator of transcription in Saccharomyces cerevisiae , 1995, Molecular and cellular biology.

[34]  G. Roeder,et al.  Recombination-stimulating sequences in yeast ribosomal DNA correspond to sequences regulating transcription by RNA polymerase I , 1987, Cell.

[35]  J. Manley,et al.  RNA polymerase II and the integration of nuclear events. , 2000, Genes & development.

[36]  H. Ikeda,et al.  Transcription promotes recA-independent recombination mediated by DNA-dependent RNA polymerase in Escherichia coli. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[37]  H. Klein,et al.  HPR1, a novel yeast gene that prevents intrachromosomal excision recombination, shows carboxy-terminal homology to the Saccharomyces cerevisiae TOP1 gene , 1990, Molecular and cellular biology.

[38]  M. Christman,et al.  Isolation of mutants of Saccharomyces cerevisiae requiring DNA topoisomerase I. , 1995, Genetics.

[39]  J. Griffin,et al.  Induction of RNA-stabilized DMA conformers by transcription of an immunoglobulin switch region , 1990, Nature.

[40]  Gerald R. Fink,et al.  Mitotic recombination in the rDNA of S. cerevisiae is suppressed by the combined action of DNA topoisomerases I and II , 1988, Cell.

[41]  G. Orphanides,et al.  A Unified Theory of Gene Expression , 2002, Cell.

[42]  S. Chávez,et al.  Hpr1 Is Preferentially Required for Transcription of Either Long or G+C-Rich DNA Sequences in Saccharomyces cerevisiae , 2001, Molecular and Cellular Biology.

[43]  R. K. Herman,et al.  Effect of Gene Induction on the Rate of Mutagenesis by ICR-191 in Escherichia coli , 1971, Journal of bacteriology.

[44]  J. Nickoloff.  Transcription enhances intrachromosomal homologous recombination in mammalian cells , 1992, Molecular and cellular biology.

[45]  Charles Fortin,et al.  The problem of hypernegative supercoiling and R-loop formation in transcription. , 2003, Frontiers in bioscience : a journal and virtual library.

[46]  B. Michel,et al.  Transcription‐induced deletions in Escherichia coli plasmids , 1995, Molecular microbiology.

[47]  M. Gallardo,et al.  A new hyperrecombination mutation identifies a novel yeast gene, THP1, connecting transcription elongation with mitotic recombination. , 2001, Genetics.

[48]  Daniel Zenklusen,et al.  Stable mRNP Formation and Export Require Cotranscriptional Recruitment of the mRNA Export Factors Yra1p and Sub2p by Hpr1p , 2002, Molecular and Cellular Biology.

[49]  DNA Double-Strand Breaks , 2002, The Journal of Experimental Medicine.

[50]  H. Fan,et al.  Characterization of mutations that suppress the temperature-sensitive growth of the hpr1 delta mutant of Saccharomyces cerevisiae. , 1994, Genetics.