Researchers reveal the mechanism of DNA repair
(Nanowerk News) A new study adds to a radically new emerging picture of how bacterial cells continually repair damaged parts of their DNA.
Published in a journal Cell (“RNA Polymerase Drives DNA Repair Ribonucleotide Excision in E. coli“), the report describes the molecular mechanisms behind DNA repair pathways that counteract the incorrect inclusion of certain types of molecular building blocks, ribonucleotides, into the genetic code.
Such errors occur frequently in the code-copying process in bacteria and other organisms. Given that ribonucleotide fusion errors can result in harmful DNA code changes (mutations) and DNA breaks, all organisms have evolved to possess a DNA repair pathway called ribonucleotide excision repair (RER) that rapidly corrects such errors.
Last year a team led by Evgeny Nudler, PhD, Professor Julie Wilson Anderson in the Department of Molecular Biochemistry and Pharmacology at NYU Langone Health, published two analyzes of DNA repair in living organisms. E. coli cell. They found that most repairs of certain types of DNA damage (large lesions), such as those caused by UV radiation, can occur because the damaged part of the code is first identified by a protein machine called RNA polymerase. RNA polymerase drives the DNA chain, reading the DNA “letter” code as it transcribes instructions into RNA molecules, which then direct the formation of proteins.
Nudler and co-workers found that during this transcription process, RNA polymerase also locates DNA lesions, and then serves as a platform for the assembly of a DNA repair machine called the nucleotide excision repair (NER) complex. NER then takes the faulty DNA it finds and replaces it with an accurate copy. Without the action of RNA polymerase, little, if any, NER occurs in living bacteria.
Now new studies in Cells provide the first evidence that, like in the NER pathway, RER is intimately linked to transcription. The study authors found evidence that the key enzyme involved in RER, RNaseHII, also cooperates with RNA polymerase when scanning for faulty ribonucleotides in the DNA chain of living bacterial cells.
“Our results continue to inspire a rethink of certain fundamental principles in the field of DNA repair,” said Nudler, also a researcher at the Howard Hughes Medical Institute. “Going forward, our team plans to investigate whether RNA polymerase scans DNA for all kinds of problems and triggers genome-wide repair, not only in bacteria, but also in human cells.”
Ribonucleotides (the building blocks of RNA) and deoxyribonucleotides (components of DNA) are related compounds. When cells copy and build DNA chains in bacterial cells, they often mistakenly insert ribonucleotides into the DNA chains instead of deoxyribonucleotides because they only differ by one oxygen atom, the study authors said. In bacterial cells, DNA polymerase III is known to make about 2,000 of these errors each time it copies the cell’s genetic material. In order to preserve genome integrity, most of the misplaced ribonucleotides are eliminated via the RER pathway, but the key question is how RNaseHII finds relatively rare ribonucleotide lesions amidst the “sea” of intact cellular DNA code so quickly.
As they did in their 2022 study, the researchers used quantitative mass spectrometry and in vivo protein-protein crosslinking to map the distances between chemically linked proteins, and thereby determine the main surfaces of RNaseHII and RNA polymerase as they interact in the bacterial cell. life. In this way they determined that most of the RNaseHII molecules couple to RNA polymerase.
In addition, they used cryogenic electron microscopy (CryoEM) to capture the high-resolution structures of RNaseHII bound to RNA polymerase to reveal the protein-protein interactions that define the RER complex. Furthermore, structure-guided genetic experiments that attenuate the RNA polymerase/RNaseHII interaction compromise the RER.
“This work supports a model in which RNaseHII scans DNA for misplaced ribonucleotides by driving RNA polymerase as it moves along DNA,” said first study author Zhitai Hao, a post-doctoral scholar in Nudler’s lab. “This work is critical to our basic understanding of the DNA repair process and has far-reaching clinical implications.”