A new study demonstrates that a method may identify, for the first time, the frequency and precise location of a molecular occurrence known as "backtracking" across any species' genetic material (genome).
The study was published in the journal Molecular Cell and lends credence to the hypothesis that backtracking, which affects thousands of human genes, many of which are involved in fundamental biological processes like cell division and development in the womb, represents a common mode of gene regulation.
The study examines genes, which consist of long strands of DNA composed of molecular "bases" sequenced in a precise order to encode the genetic instructions essential for the development and functioning of all living organisms. The researchers leading this investigation are from NYU Grossman School of Medicine.
Transcription, the initial stage of gene expression in both humans and bacteria, is carried out by RNA polymerase II, a protein "machine" that moves down the DNA chain and reads genetic instructions in one direction.
In a 1997 study, Evgeny A. Nudler, Ph.D., and associates demonstrated that RNA polymerase occasionally moves backward along the strand it is reading, a behavior they dubbed "backtracking."
Since then, research has revealed that backtracking occasionally occurs in living cells, either shortly after RNA polymerase synthesizes RNA or when it comes into contact with damaged DNA and needs to make space for incoming repair enzymes.
Further research indicated that the machinery responsible for backsliding and repair needed to evaporate rapidly to avoid colliding with DNA polymerase and breaking DNA chains, which would result in cell death.
Recently, a study by the NYU Langone Health team led by Dr Nudler demonstrates that backtracking events can be directly detected using a new approach called long-range cleavage sequencing (LORAX-seq).
The new method demonstrated that many such occurrences migrate backward farther than previously assumed and, in doing so, endure longer. This complements previous approaches that were indirect or limited.
Furthermore, the data imply that persistent backtracking has purposes far beyond DNA repair, occurs often across genomes, and occurs more frequently near specific gene types.
The surprising stability of backtracking at longer distances makes it likely that it represents a ubiquitous form of genetic regulation in species from bacteria to humans, and if further work expands our findings to different developmental programs and pathological conditions, backtracking may be akin to epigenetics, the discovery of which revealed a surprising new layer of gene regulation without changing the DNA code.”
Evgeny A. Nudler, Study Senior Author, and Julie Wilson Anderson Professor, Department of Biochemistry and Molecular Pharmacology, NYU Langone Health
Central to Life?
The enzyme RNA polymerase II converts DNA code into RNA, a similar substance that instructs the synthesis of proteins. To accomplish this, the complex travels down DNA chains in one direction, but occasionally it turns around.
Previous research has demonstrated that when RNA polymerase II reverses course, the tip of the RNA chain it has been synthesizing according to the DNA code is forced out (extruded) from its internal channel.
Since extended backtracking might lead to harmful collisions, it is believed that transcription is rapidly resumed by the transcription factor IIS (TFIIS), which encourages the cleavage of the extruded, "backtracked" RNA. RNA polymerase II can now resume reading forward codes because of this.
However, prior research has demonstrated that polymerase can connect to the channel through which it is extruded and retain the backtracked RNA in place for a longer period when it backtracks beyond a specific distance (such as 20 nucleobase DNA building blocks).
TFIIS-driven cleavage is less likely to save locked, backtracked complexes and is more likely to cause delayed transcription of the underlying gene. This gave rise to the hypothesis that backtracking may regulate the activity of genes as a primary regulatory mechanism and also play a crucial role in DNA repair processes.
The researchers conclude that TFIIS probably exists in living cells at low concentrations where it competes with hundreds of other proteins to access and remove RNA that has been sidetracked, allowing transcription to proceed.
Rather than using competing proteins, the scientists in the current study employed a high concentration of purified TFIIS to precisely remove any backtracked RNA fragment found anywhere in a cell's genetic code. This allowed tools that decipher code sequences and reveal clues about their locations and functions to access the cutout bits.
Additionally, the study team discovered that genes regulating histones protein "spools" that DNA chains wind around in the chromatin that arranges gene expression are extremely prone to repeatedly going backward.
According to the authors ' theory, the degree to which this occurs, together with associated modifications in the transcription of certain genes, may regulate the time of large-scale histone accumulation required for chromatin reconstruction during cell division.
Furthermore, they imply that repeated backtracking might affect the prompt transcription of genes essential for tissue growth.
Along with its potentially useful functions, persistent backtracking could also result in DNA damage and other genetic malfunctions that contribute to disease, and we speculate that the measurement of backtracking in the context of aging or cancer, for instance, may help us understand why malfunctions occur in the cell stress response and cell replication, and to suggest new treatment approaches.”
Kevin Yang, Study First Author and Graduate Student, NYU Langone Health
Yang, K. B., et.al., (2024). Persistence of backtracking by human RNA polymerase II. Molecular Cell. doi.org/10.1016/j.molcel.2024.01.019