A model for transcription-dependent R-loop formation at double-stranded DNA breaks: Implications for their detection and biological effects.

R-loops are structures containing an RNA-DNA duplex and an unpaired DNA strand. During R-loop formation an RNA strand invades the DNA duplex, displacing the homologous DNA strand and binding the complementary DNA strand. Here we analyze a model for transcription-dependent R-loop formation at double-stranded DNA breaks (DSBs).

Evelyn M. Witkin (1921-2023).

Pioneer of cell mutagenesis and DNA repair research.

Unbalanced Growth, the DNA Replication Cycle and Discovery of Repair Replication.

This article recounts my graduate research at Yale University (1954-1958) on unbalanced growth in during thymine deprivation or following ultraviolet (UV) irradiation, with early evidence for the repair of UV-induced DNA damage. Follow-up studies in Copenhagen (1958-1960) in the laboratory of Ole Maaløe led to my discovery that the DNA replication cycle can be synchronized by inhibiting protein and RNA syntheses and that an RNA synthesis step is essential for initiation of the cycle, but not for its completion. This work set the stage for my subsequent research at Stanford University, where the repair replication of damaged DNA was documented, to provide compelling evidence for an excision-repair pathway.

Topology and kinetics of R-loop formation.

R-loops are structures containing an RNA-DNA duplex and an unpaired DNA strand. They can be formed upon "invasion" of an RNA strand into a DNA duplex, during which the RNA displaces the homologous DNA strand and binds the complementary strand. R-loops have many significant beneficial or deleterious biological effects, so it is important to understand the mechanisms for their generation and processing.

Transcription Inhibition by PNA-Induced R-Loops.

R-loops are structures consisting of an RNA-DNA duplex and an unpaired DNA strand. They can form during transcription upon nascent RNA "threadback" invasion into the DNA duplex to displace the non-template DNA strand. R-loops occur naturally in all kingdoms of life, and they have multiple biological effects.

Mechanistic understanding of cellular responses to genomic stress.

Within the past half century we have learned of multiple pathways for repairing damaged DNA, based upon the intrinsic redundancy of information in its complementary double strands. Mechanistic details of these pathways have provided insights into environmental and endogenous threats to genomic stability. Studies on bacterial responses to ultraviolet light led to the discovery of excision repair, as well as the inducible SOS response to DNA damage.

R-loop generation during transcription: Formation, processing and cellular outcomes.

R-loops are structures consisting of an RNA-DNA duplex and an unpaired DNA strand. They can form during transcription upon nascent RNA "threadback" invasion into the DNA duplex to displace the non-template strand. Although R-loops occur naturally in all kingdoms of life and serve regulatory roles, they are often deleterious and can cause genomic instability.

A novel mode for transcription inhibition mediated by PNA-induced R-loops with a model in vitro system.

The selective inhibition of transcription of a chosen gene by an artificial agent has numerous applications. Usually, these agents are designed to bind a specific nucleotide sequence in the promoter or within the transcribed region of the chosen gene. However, since optimal binding sites might not exist within the gene, it is of interest to explore the possibility of transcription inhibition when the agent is designed to bind at other locations.

Strong transcription blockage mediated by R-loop formation within a G-rich homopurine-homopyrimidine sequence localized in the vicinity of the promoter.

Guanine-rich (G-rich) homopurine-homopyrimidine nucleotide sequences can block transcription with an efficiency that depends upon their orientation, composition and length, as well as the presence of negative supercoiling or breaks in the non-template DNA strand. We report that a G-rich sequence in the non-template strand reduces the yield of T7 RNA polymerase transcription by more than an order of magnitude when positioned close (9 bp) to the promoter, in comparison to that for a distal (∼250 bp) location of the same sequence. This transcription blockage is much less pronounced for a C-rich sequence, and is not significant for an A-rich sequence.

Modulation of Cytotoxicity by Transcription-Coupled Nucleotide Excision Repair Is Independent of the Requirement for Bioactivation of Acylfulvene.

Bioactivation as well as DNA repair affects the susceptibility of cancer cells to the action of DNA-alkylating chemotherapeutic drugs. However, information is limited with regard to the relative contributions of these processes to the biological outcome of metabolically activated DNA alkylating agents. We evaluated the influence of cellular bioactivation capacity and DNA repair on cytotoxicity of the DNA alkylating agent acylfulvene (AF).

When transcription goes on Holliday: Double Holliday junctions block RNA polymerase II transcription in vitro.

Non-canonical DNA structures can obstruct transcription. This transcription blockage could have various biological consequences, including genomic instability and gratuitous transcription-coupled repair. Among potential structures causing transcription blockage are Holliday junctions (HJs), which can be generated as intermediates in homologous recombination or during processing of stalled replication forks.

Mutational Strand Asymmetries in Cancer Genomes Reveal Mechanisms of DNA Damage and Repair.

Mutational processes constantly shape the somatic genome, leading to immunity, aging, cancer, and other diseases. When cancer is the outcome, we are afforded a glimpse into these processes by the clonal expansion of the malignant cell. Here, we characterize a less explored layer of the mutational landscape of cancer: mutational asymmetries between the two DNA strands.

Historical perspective on the DNA damage response.

The DNA damage response (DDR) has been broadly defined as a complex network of cellular pathways that cooperate to sense and repair lesions in DNA. Multiple types of DNA damage, some natural DNA sequences, nucleotide pool deficiencies and collisions with transcription complexes can cause replication arrest to elicit the DDR. However, in practice, the term DDR as applied to eukaryotic/mammalian cells often refers more specifically to pathways involving the activation of the ATM (ataxia-telangiectasia mutated) and ATR (ATM-Rad3-related) kinases in response to double-strand breaks or arrested replication forks, respectively.

Photobiological Origins of the Field of Genomic Maintenance.

Although sunlight is essential for life on earth, the ultraviolet (UV) wavelengths in its spectrum constitute a major threat to life. Various cellular responses have evolved to deal with the damage inflicted in DNA by UV, and the study of these responses in model systems has spawned the burgeoning field of DNA repair. Although we now know of many types of deleterious alterations in DNA, the approaches for studying them and the early mechanistic insights have come in large part from pioneering research on the processing of UV-induced bipyrimidine photoproducts in bacteria.

Photosensitive human syndromes.

Photosensitivity in humans can result from defects in repair of light-induced DNA lesions, from photoactivation of chemicals (including certain medications) with sunlight to produce toxic mediators, and by immune reactions to sunlight exposures. Deficiencies in DNA repair and the processing of damaged DNA during replication and transcription may result in mutations and genomic instability. We will review current understanding of photosensitivity to short wavelength ultraviolet light (UV) due to genetic defects in particular DNA repair pathways; deficiencies in some are characterized by an extremely high incidence of cancer in sun-exposed tissues, while in others no cancers have been reported.

Thymineless Death Lives On: New Insights into a Classic Phenomenon.

The primary mechanisms by which bacteria lose viability when deprived of thymine have been elusive for over half a century. Early research focused on stalled replication forks and the deleterious effects of uracil incorporation into DNA from thymidine-deficient nucleotide pools. The initiation of the replication cycle and origin-proximal DNA degradation during thymine starvation have now been quantified via whole-genome microarrays and other approaches.