ABSTRACT
The MR (Mre11 nuclease and Rad50 ABC ATPase) complex is an evolutionarily conserved sensor for DNA double-strand breaks, highly genotoxic lesions linked to cancer development. MR can recognize and process DNA ends even if they are blocked and misfolded. To reveal its mechanism, we determined the crystal structure of the catalytic head of Thermotoga maritima MR and analyzed ATP-dependent conformational changes. MR adopts an open form with a central Mre11 nuclease dimer and two peripheral Rad50 molecules, a form suited for sensing obstructed breaks. The Mre11 C-terminal helix-loop-helix domain binds Rad50 and attaches flexibly to the nuclease domain, enabling large conformational changes. ATP binding to the two Rad50 subunits induces a rotation of the Mre11 helix-loop-helix and Rad50 coiled-coil domains, creating a clamp conformation with increased DNA-binding activity. The results suggest that MR is an ATP-controlled transient molecular clamp at DNA double-strand breaks.
Subject(s)
Adenosine Triphosphate/metabolism , Bacterial Proteins/chemistry , DNA Repair Enzymes/chemistry , DNA Repair , DNA-Binding Proteins/chemistry , Thermotoga maritima/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Crystallography, X-Ray , DNA Breaks, Double-Stranded , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/metabolism , Exodeoxyribonucleases/chemistry , Exodeoxyribonucleases/metabolism , Models, Molecular , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Scattering, Small Angle , Thermotoga maritima/metabolism , X-Ray DiffractionABSTRACT
Multiple DNA-associated processes such as DNA repair, replication, and recombination are crucial for the maintenance of genome integrity. Here, we show a novel interaction between the transcription elongation factor Bur1-Bur2 and replication protein A (RPA), the eukaryotic single-stranded DNA-binding protein with functions in DNA repair, recombination, and replication. Bur1 interacted via its C-terminal domain with RPA, and bur1-ΔC mutants showed a deregulated DNA damage response accompanied by increased sensitivity to DNA damage and replication stress as well as increased levels of persisting Rad52 foci. Interestingly, the DNA damage sensitivity of an rfa1 mutant was suppressed by bur1 mutation, further underscoring a functional link between these two protein complexes. The transcription elongation factor Bur1-Bur2 interacts with RPA and maintains genome integrity during DNA replication stress.
Subject(s)
Cyclin-Dependent Kinases/chemistry , Cyclins/chemistry , Mutation , Replication Protein A/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Alleles , DNA Damage , DNA Replication , Genome , Genome-Wide Association Study , Microscopy, Fluorescence/methods , Oligonucleotide Array Sequence Analysis , Protein Interaction Mapping , Protein Structure, Tertiary , Recombination, Genetic , TemperatureABSTRACT
The conserved TREX complex couples transcription to nuclear mRNA export. Here, we report that the uncharacterized open reading frame YOR166c genetically interacts with TREX complex components and encodes a novel protein named Swt1 for "synthetically lethal with TREX." Co-immunoprecipitation experiments show that Swt1 also interacts with the TREX complex biochemically. Consistent with a potential role in transcription as suggested by its interaction with TREX, Swt1 localizes mainly to the nucleus. Importantly, deletion of Swt1 leads to decreased transcription. Taken together, these data suggest that Swt1 functions in gene expression in conjunction with the TREX complex.
