Project description:The structure of broken DNA ends is a critical determinant of the pathway used for DNA double strand break (DSB) repair. Here, we develop an approach, hairpin capture of DNA end structures (HCoDES), which elucidates chromosomal DNA end structures at single nucleotide resolution. HCoDES defines structures of physiologic DSBs generated by the RAG endonuclease, as well as those generated by nucleases widely used for genome editing. Analysis of G1-phase cells deficient in H2AX or 53BP1 reveals DNA ends that are frequently resected to form long single-stranded overhangs that can be repaired by mutagenic pathways. In addition to 3’ overhangs, many of these DNA ends unexpectedly form long 5’ single-stranded overhangs. The divergence in DNA end structures resolved by HCoDES suggests that H2AX and 53BP1 may have distinct activities in end protection. Thus, the high-resolution end structures obtained by HCoDES identify new features of DNA end processing during DSB repair. Single stranded DNA ligation of genomic DNA isolated from G1 arrested LigaseIV-/-, LigaseIV-/- 53BP1-/- and LigaseIV-/- H2AX-/- Abelson pre-B cells harboring site specific DSBs generated by the RAG recombinase, Cas9 endonuclease or Zinc Finger Endonuclease.
Project description:The structure of broken DNA ends is a critical determinant of the pathway used for DNA double strand break (DSB) repair. Here, we develop an approach, hairpin capture of DNA end structures (HCoDES), which elucidates chromosomal DNA end structures at single nucleotide resolution. HCoDES defines structures of physiologic DSBs generated by the RAG endonuclease, as well as those generated by nucleases widely used for genome editing. Analysis of G1-phase cells deficient in H2AX or 53BP1 reveals DNA ends that are frequently resected to form long single-stranded overhangs that can be repaired by mutagenic pathways. In addition to 3’ overhangs, many of these DNA ends unexpectedly form long 5’ single-stranded overhangs. The divergence in DNA end structures resolved by HCoDES suggests that H2AX and 53BP1 may have distinct activities in end protection. Thus, the high-resolution end structures obtained by HCoDES identify new features of DNA end processing during DSB repair.
Project description:Mapping ultra high resolution of Brachyury:DNA interaction would provide us with valuable new mechanistic insights into complex molecular transactions at Brachyury-bound enhancers.
Project description:Transcription termination in bacteria can occur either via Rho-dependent or independent (intrinsic) mechanisms. Intrinsic terminators are composed of a stem-loop RNA structure followed by a uridine stretch and are known to terminate in a precise manner. In contrast, Rho-dependent terminators have more loosely defined characteristics and are thought to terminate in a diffuse manner. While transcripts ending in an intrinsic terminator are protected from 3’-5’ exonuclease digestion due to the stem-loop structure of the terminator, it remains unclear what protects Rho-dependent transcripts from being degraded. In this study, we mapped the exact steady-state RNA 3’ ends of hundreds of E. coli genes terminated either by Rho-dependent or independent mechanisms. We found that transcripts generated from Rho-dependent termination have precise 3’-ends at steady state. These termini were localized immediately downstream of energetically stable stem-loop structures, which were not followed by uridine rich sequences. We provide evidence that these structures protect Rho-dependent transcripts from 3’-5’ exonucleases such as PNPase and RNase II, and present data localizing the Rho-utilization (rut) sites immediately downstream of these protective structures. This study represents the first extensive in-vivo map of exact RNA 3’-ends of Rho-dependent transcripts in E. coli.
Project description:Mapping ultra high resolution of Brachyury:DNA interaction would provide us with valuable new mechanistic insights into complex molecular transactions at Brachyury-bound enhancers. Embryonic stem cells were differentiated into Brachyury-positive mesoendoderm cells. And, ChIP-exo experiment was then performed to identify detailed Brachyury-DNA binding profiles.