During the essential processes of DNA replication and transcription, RNA-DNA hybrid intermediates are formed that pose significant risks to genome integrity when left unresolved. To manage RNA-DNA hybrids, all cells rely on RNase H family enzymes that specifically cleave the RNA portion of the many different types of hybrids that form in vivo. Recent experimental advances have provided new insight into how RNA-DNA hybrids form and the consequences to genome integrity that ensue when persistent hybrids remain unresolved. Here we review the types of RNA-DNA hybrids, including R-loops, RNA primers, and ribonucleotide misincorporations, that form during DNA replication and transcription and discuss how each type of hybrid can contribute to genome instability in bacteria. Further, we discuss how bacterial RNase HI, HII, and HIII and bacterial FEN enzymes contribute to genome maintenance through the resolution of hybrids.
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http://dx.doi.org/10.1146/annurev-micro-102521-014450 | DOI Listing |
Spatially mapping the transcriptome and proteome in the same tissue section can significantly advance our understanding of heterogeneous cellular processes and connect cell type to function. Here, we present Deterministic Barcoding in Tissue sequencing plus (DBiTplus), an integrative multi-modality spatial omics approach that combines sequencing-based spatial transcriptomics and image-based spatial protein profiling on the same tissue section to enable both single-cell resolution cell typing and genome-scale interrogation of biological pathways. DBiTplus begins with reverse transcription for cDNA synthesis, microfluidic delivery of DNA oligos for spatial barcoding, retrieval of barcoded cDNA using RNaseH, an enzyme that selectively degrades RNA in an RNA-DNA hybrid, preserving the intact tissue section for high-plex protein imaging with CODEX.
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December 2024
Department of Gynecology, The Second Affiliated Hospital, Zhejiang University School of Medicine, 310058 Hangzhou, China; Department of Cell Biology, Zhejiang University School of Medicine, 310058 Hangzhou, China. Electronic address:
Stalled replication forks, susceptible to nucleolytic threats, necessitate protective mechanisms involving pivotal factors such as the tumor suppressors BRCA1 and BRCA2. Here, we demonstrate that, upon replication stress, RNA polymerase II (RNAPII) is recruited to stalled forks, actively promoting the transient formation of RNA-DNA hybrids. These hybrids act as safeguards, preventing premature engagement by the DNA2 nuclease and uncontrolled DNA2-mediated degradation of nascent DNA.
View Article and Find Full Text PDFMol Cell
December 2024
Department of Radiation Medicine, School of Basic Medical Sciences, Peking University International Cancer Institute, Institute of Advanced Clinical Medicine, State Key Laboratory of Molecular Oncology, Peking University Health Science Center, Beijing 100191, China; Department of Gastrointestinal Translational Research, Peking University Cancer Hospital, Beijing 100142, China. Electronic address:
Safeguarding replication fork stability in transcriptionally active regions is crucial for precise DNA replication and mutation prevention. Here, we discover the pervasive existence of replication fork-associated RNA-DNA hybrids (RF-RDs) in transcriptionally active regions of human cells. These hybrids function as protective barriers, preventing DNA2-mediated nascent DNA degradation and replication fork collapse under replication stress.
View Article and Find Full Text PDFFEBS Lett
December 2024
Department of Biochemistry and Molecular Biology, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, USA.
Genome maintenance is essential for the integrity of the genetic blueprint, of which only a small fraction is transcribed in higher eukaryotes. DNA lesions occurring in the transcribed genome trigger transcription pausing and transcription-coupled DNA repair. There are two major transcription-coupled DNA repair pathways.
View Article and Find Full Text PDFNucleic Acids Res
December 2024
Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA.
RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ∼20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes.
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