R-loops are three-stranded structures generated by annealing of nascent transcripts to the template DNA strand, leaving the non-template DNA strand exposed as a single-stranded loop. Although R-loops play important roles in physiological processes such as regulation of gene expression, mitochondrial DNA replication, or immunoglobulin class switch recombination, dysregulation of the R-loop metabolism poses a threat to the stability of the genome. A previous study in yeast has shown that the homologous recombination machinery contributes to the formation of R-loops and associated chromosome instability. On the contrary, here, we demonstrate that depletion of the key homologous recombination factor, RAD51, as well as RAD51 inhibition by the B02 inhibitor did not prevent R-loop formation induced by the inhibition of spliceosome assembly in human cells. However, we noticed that treatment of cells with B02 resulted in RAD51-dependent accumulation of R-loops in an early G1 phase of the cell cycle accompanied by a decrease in the levels of chromatin-bound ORC2 protein, a component of the pre-replication complex, and an increase in DNA synthesis. Our results suggest that B02-induced R-loops might cause a premature origin firing.

• Keywords
• B02 inhibitor, G1 phase of the cell cycle, R-loop, RAD51, origin of replication, pre-replication complex,
• MeSH
• Chromosomal Instability drug effects   MeSH
• DNA biosynthesis   MeSH
• G1 Phase drug effects   MeSH
• Enzyme Inhibitors pharmacology   MeSH
• Origin Recognition Complex metabolism   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• R-Loop Structures * MeSH
• Rad51 Recombinase * antagonists & inhibitors   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA MeSH
• Enzyme Inhibitors MeSH
• Origin Recognition Complex MeSH
• ORC2 protein, human MeSH Browser
• RAD51 protein, human MeSH Browser
• Rad51 Recombinase * MeSH

RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51-ssDNA filaments. RECQ5 interacts with RAD51 through protein-protein contacts, and disruption of this interface through a RECQ5-F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51-K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51-I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.

• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Point Mutation MeSH
• RecQ Helicases genetics   metabolism   ultrastructure   MeSH
• Homologous Recombination * MeSH
• Hydrolysis MeSH
• DNA, Single-Stranded metabolism   ultrastructure   MeSH
• Kinetics MeSH
• Humans MeSH
• Microscopy, Atomic Force MeSH
• Mutation, Missense MeSH
• Molecular Motor Proteins metabolism   ultrastructure   MeSH
• Recombinant Fusion Proteins metabolism   MeSH
• Recombinant Proteins metabolism   MeSH
• Rad51 Recombinase genetics   metabolism   MeSH
• Replication Protein A metabolism   MeSH
• Substrate Specificity MeSH
• Single Molecule Imaging * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• RecQ Helicases MeSH
• DNA, Single-Stranded MeSH
• Molecular Motor Proteins MeSH
• RAD51 protein, human MeSH Browser
• RECQL5 protein, human MeSH Browser
• Recombinant Fusion Proteins MeSH
• Recombinant Proteins MeSH
• Rad51 Recombinase MeSH
• Replication Protein A MeSH
• RPA1 protein, human MeSH Browser

RECQ5 belongs to the RecQ family of DNA helicases. It is conserved from Drosophila to humans and its deficiency results in genomic instability and cancer susceptibility in mice. Human RECQ5 is known for its ability to regulate homologous recombination by disrupting RAD51 nucleoprotein filaments. It also binds to RNA polymerase II (RNAPII) and negatively regulates transcript elongation by RNAPII. Here, we summarize recent studies implicating RECQ5 in the prevention and resolution of transcription-replication conflicts, a major intrinsic source of genomic instability during cancer development.

• Keywords
• DNA repair, R-loops, RECQ5, genomic instability, replication stress, transcription-replication conflicts,
• MeSH
• DNA genetics   metabolism   MeSH
• Transcription, Genetic genetics   MeSH
• RecQ Helicases genetics   metabolism   physiology   MeSH
• Humans MeSH
• Genomic Instability MeSH
• DNA Replication MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• DNA MeSH
• RecQ Helicases MeSH