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Crystal structure of E. coli RecE protein reveals a toroidal tetramer for processing double-stranded DNA breaks.

ABSTRACT: Escherichia coli RecE protein is part of the classical RecET recombination system that has recently been used in powerful new methods for genetic engineering. RecE binds to free double-stranded DNA (dsDNA) ends and processively digests the 5'-ended strand to form 5'-mononucleotides and a 3'-overhang that is a substrate for single strand annealing promoted by RecT. Here, we report the crystal structure of the C-terminal nuclease domain of RecE at 2.8 A resolution. RecE forms a toroidal tetramer with a central tapered channel that is wide enough to bind dsDNA at one end, but is partially plugged at the other end by the C-terminal segment of the protein. Four narrow tunnels, one within each subunit of the tetramer, lead from the central channel to the four active sites, which lie about 15 A from the channel. The structure, combined with mutational studies, suggests a mechanism in which dsDNA enters through the open end of the central channel, the 5'-ended strand passes through a tunnel to access one of the four active sites, and the 3'-ended strand passes through the plugged end of the channel at the back of the tetramer.

SUBMITTER: Zhang J

PROVIDER: S-EPMC2688778 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Crystal structure of E. coli RecE protein reveals a toroidal tetramer for processing double-stranded DNA breaks.

Zhang Jinjin J Xing Xu X Herr Andrew B AB Bell Charles E CE

Structure (London, England : 1993) 20090501 5

Escherichia coli RecE protein is part of the classical RecET recombination system that has recently been used in powerful new methods for genetic engineering. RecE binds to free double-stranded DNA (dsDNA) ends and processively digests the 5'-ended strand to form 5'-mononucleotides and a 3'-overhang that is a substrate for single strand annealing promoted by RecT. Here, we report the crystal structure of the C-terminal nuclease domain of RecE at 2.8 A resolution. RecE forms a toroidal tetramer w ...[more]

PMID: 19446525

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Project description:ABSTRACT Aims: Double-stranded breaks (DSBs) cause genomic instability, a hallmark of aging, and activate the DNA damage response (DDR) and the cytosolic DNA-sensing proteins (CDSP) pathways. The activated pathways induce cell cycle arrest, inflammation, senescence, cell death, fibrosis, and organ dysfunction. The purpose of the study was to identify and characterize genome-wide DSBs at the nucleotide resolution in adult cardiac myocytes. Methods and Results: We identified the genome-wide DSBs in the genomes of ~ 6 million adult cardiac myocytes (1 million per sample) in the 3 wild-type and 3 myocyte-specific LMNA-deficient mice (Myh6-Cre:LmnaF/F) mice by END-Sequencing. We identified 6,774 differential DSBs between the wild-type and Myh6-Cre:LmnaF/F cardiac myocytes, which, except for 8, were exclusive to the Myh6-Cre:LmnaF/F genotype. The differential DSBs were enriched in the gene regions, transcription initiation sites, cardiac transcription factor motifs, and at the G quadruplex forming structures, suggesting the involvement of the transcriptional stress. Because LMNA regulates transcription, we performed a CUT&RUN assay with an anti-LMNA antibody (N=5) to test the role of transcriptional stress in an increased prevalence of DSBs. We identified an average of 818 genome-wide lamin-associated domains (LADs), which encompassed 30% of the mouse genome. LADs were associated with a 16-fold reduction in the transcript levels of protein-coding genes located at the LAD regions (N=3,975) as opposed to genes located at the non-LAD regions (N=~ 17,778). Consistent with increased transcription at the non-LAD regions DSBs were ~ 6-fold more prevalent in the non-LAD than LAD regions. Likewise, the loss of LAD was associated with a 2.3-fold higher prevalence of differential DSBs in the Myh6-Cre:LmnaF/F cardiac myocyte genomes, which were predominantly localized to the transcription start sites. Conclusions: To our knowledge, this is the first identification of the DSBs, a hallmark of aging, at the nucleotide resolution in the cardiovascular system. The findings implicate transcriptional stress in the pathogenesis of DSB and the protective role of LMNA against DSBs likely through transcriptional suppression. Given the ubiquitous nature of transcriptional stress, DSBs are expected to be pervasive and pathogenic through activation of the DDR and CDSP pathways not only in cardiovascular diseases but also in other conditions, including aging.

Dataset Information

Crystal structure of E. coli RecE protein reveals a toroidal tetramer for processing double-stranded DNA breaks.

Publications

Crystal structure of E. coli RecE protein reveals a toroidal tetramer for processing double-stranded DNA breaks.

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