User:Ileon2019/R-loop

R-loop mapping[edit]
R-loop mapping is a laboratory technique used to distinguish introns from exons in double-stranded DNA. These R-loops are visualized by electron microscopy and reveal intron regions of DNA by creating unbound loops at these regions. A common goal of R-loop mapping includes surveying the population average in order to examine the abundance and distribution of R-loops. One method used for R-loop mapping involves the use of DRIP, an S9.6 antibody-based mapping strategy specifically directed against DNA:RNA hybrids. However, there are several variations of the DRIP method used to examine R-loop mapping at different degrees of resolution and strandedness. Another R-loop mapping technique involves the brief expression of dRNase H1. This method can be induced as an alternative method to examine R-loop patterns specifically using chromatin immunoprecipitation (ChIP) at dRNA H1 binding sites. The MapR approach also uses dRNase H1 in order to target MNase to R-loops.

R-loops as genetic damage[edit]
When unscheduled R-loops form, they can cause DNA damage and genomic instability by a number of different mechanisms. Exposed single-stranded DNA can come under attack by endogenous mutagens, including DNA-modifying enzymes such as activation-induced cytidine deaminase, and can block replication forks to induce fork collapse and subsequent double-strand breaks. As well, R-loops may induce unscheduled replication by acting as a primer. A prime example would involve rnhA mutants which are capable of independently replicating their genome without regards to the oriC. Damage is commonly observed when R-loops amass and remain active. This accumulation of R-loops has been identified as “harmful R-loops”. Correspondingly, mutants involving recG gene knockdown have been observed to encode for a helicase whose activity leads to the resolution of R-loop accumulation.

Common DNA damage and genomic instability caused by R-loops have been due to defects involving cleavage, RNA export, and splicing activity during transcription. In addition to cellular processing components, instability can also be created by replication stress, induced nuclease-mediated DNA breaks, as well as collisions during transcription. This is heavily supported by the phenomenon of Ribonuclease H1 over-expression. RNase H1 has a significantly beneficial ability in which it can suppress certain genomic instability phenotypes, ultimately resolving harmful R-loops.

R-loop accumulation has been associated with a number of diseases, including amyotrophic lateral sclerosis type 4 (ALS4), ataxia oculomotor apraxia type 2 (AOA2), Aicardi–Goutières syndrome, Angelman syndrome, Prader–Willi syndrome, and cancer.