A groundbreaking study, recently published in the journal Cell, sheds new light on how bacterial cells repair damaged sections of their DNA. The research unveils the molecular mechanism underlying a DNA repair pathway that addresses the erroneous insertion of ribonucleotides, a specific type of molecular building block, into genetic codes. Such mistakes frequently occur during the code-copying process in bacteria and other organisms. Ribonucleotide misincorporation can lead to harmful changes in DNA code (mutations) and DNA breaks, prompting all organisms to evolve a DNA repair pathway called ribonucleotide excision repair (RER) to swiftly rectify these errors.
In a prior study conducted last year, a team led by Dr. Evgeny Nudler, the Julie Wilson Anderson Professor in the Department of Biochemistry and Molecular Pharmacology at NYU Langone Health, examined DNA repair in living E. coli cells. They discovered that most repair of certain types of DNA damage, such as bulky lesions caused by UV irradiation, occurs after damaged sections are identified by a protein machine known as RNA polymerase. RNA polymerase traverses the DNA chain, reading the DNA “letters” to transcribe instructions into RNA molecules that guide protein synthesis.
Nudler and his colleagues found that during the transcription process, RNA polymerase also detects DNA lesions and serves as a platform for the assembly of a DNA repair apparatus called nucleotide excision repair (NER) complex. The NER complex then removes faulty DNA segments and replaces them with accurate copies. Without the involvement of RNA polymerase, little to no NER occurs in living bacteria.
The recent study in Cell provides the first evidence that, similar to the NER pathway, RER is closely linked to transcription. The researchers discovered that the key enzyme involved in RER, called RNaseHII, collaborates with RNA polymerase as it scans DNA chains in living bacterial cells for misincorporated ribonucleotides.
“Our findings challenge existing notions in the DNA repair field,” says Nudler, who is also an investigator with the Howard Hughes Medical Institute. “In our future investigations, we aim to explore whether RNA polymerase scans DNA for various types of problems, triggering genome-wide repair not only in bacteria but also in human cells.”
Cutting edge techniques
The study authors explain that ribonucleotides, the building blocks of RNA, and deoxyribonucleotides, the components of DNA, are closely related compounds. During the process of copying and constructing DNA chains in bacterial cells, ribonucleotides are often mistakenly incorporated into DNA chains instead of deoxyribonucleotides due to their similarity, differing by only a single oxygen atom. This copying error occurs approximately 2,000 times every time DNA polymerase III replicates a cell’s genetic material in bacterial cells. To maintain the integrity of the genome, the majority of these misplaced ribonucleotides are removed through the ribonucleotide excision repair (RER) pathway. However, a significant question remained regarding how RNaseHII, the key enzyme in RER, efficiently locates the relatively rare ribonucleotide lesions among a vast “ocean” of intact DNA codes.
In their previous studies conducted in 2022, the researchers employed quantitative mass spectrometry and in vivo protein-protein crosslinking techniques to determine the distances between chemically linked proteins. This enabled them to identify the key surfaces of RNaseHII and RNA polymerase when they interact within living bacterial cells. Through these experiments, they discovered that a majority of RNaseHII molecules are associated with RNA polymerase.
Furthermore, the researchers utilized cryogenic electron microscopy (CryoEM) to capture high-resolution structures of RNaseHII bound to RNA polymerase. These structural analyses unveiled the protein-protein interactions that define the RER complex. They also performed genetic experiments guided by the obtained structures, weakening the interaction between RNA polymerase and RNaseHII, which consequently compromised the efficiency of RER.
Based on their findings, the researchers propose a model in which RNaseHII scans DNA for misplaced ribonucleotides by riding along with RNA polymerase as it moves along the DNA chain. Zhitai Hao, the first author of the study and a post-doctoral scholar in Nudler’s lab, emphasizes the significance of this work in enhancing our fundamental understanding of the DNA repair process and its potential clinical implications.
Source: NYU Langone Health