Summary of Three Papers
Human mitochondria DNA is characterized by circular double-stranded molecules that are separable through the process of configuration density. The comprehension of the various roles and structures of the human mitochondrion DNA is crucial in this field. As a result, several studies have been conducted to understand replication, initiation, and DNA repair, among other essential human mitochondrion roles and structures. Authored by Posse et al., (2019), paper I focusses on the formation of stable R-loops from Ribonuclease H1 (RNase H1), therefore directly influencing DNA initiation replication of human mitochondria. Paper II highlights the primer's formation at the mitochondrion origin of light-strand DNA synthesis (OriL), which is crucial for maintaining mitochondrion DNA. Paper II's core aim is to define the role of RNase H1 in primer removal and comprehend the consequences of mutations (Al-Behadili et al., 2018). Paper III investigates the role of EXOG, which is primarily to process short DNA substrates produced during replication and repair, and its ability to work with RNase H1 in the removal of the primer. This paper summarizes the three papers whose studies are centered on human mitochondrion and includes each study's aim, significant findings, and importance.
The formation of R-loops is a series of processes that begin with RNA primers for initiation at OriH, following the LSP transcription. Based on vivo analysis, the primer synthesis becomes abolished, resulting in the switch to full-length transcription below the LSP with three sequence blocks, including CSBI-III, RNA, and DNA transitions. The newly transcribed RNA H-strands remain in the CSB region, resulting in stable R-loops resistant to treatments RNase A and RNase T1. Furthermore, in vivo, RNase H1 is essential for the degradation of R-loops. Besides, the depletion of RNase H1 results in low levels of mitochondrion DNA, hence the suggestion that RNase H1 is important in mitochondrion DNA replication. Based on the vivo findings, there is the possibility that RNase H1 plays a significant role in R-loop formation and formation of the primer. Furthermore, the characteristics of RNase H1, such as its capability to cleave on lengthy RNA-DNA hybrids, to cleave on Oka-zaki fragment structures, and its involvement in the pre-rRNA by interaction with mitochondrion protein P32 enhances its enzymatic activity (Posse et al., 2019). To confirm the possibility of RNase H1 in R-loop formation, the study investigates the process in vitro instead of previously done work based on vivo analysis.
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In vitro, the scholars used pure mitochondrion proteins from human beings and highly coiled plasmids with LSP at the CSB1-III region. Following the effects, all transcriptions were terminated prematurely at CSBIII and reduced upon the addition of TEFM. The experiment findings reveal R-loops' formation in the LSP to CSBIII region and their stability characteristics shown when TEFM was added during transcription. Further, the paper investigates whether RNase H1 can process R-loops and found out that the enzyme degraded them depending on the concentration level, resulting in short RNAs. Their findings are in alignment with previously done studies that suggested that R-loops can be processed from RNase H1.
In addition to their formation, R-loops can also be used as primers for mitochondrion DNA synthesis initiation. The scholars randomly selected RNA primers to initiate DNA synthesis in negatively coiled dsDNA to investigate this role. Following the investigation, DNA products were observed, hence the conclusion that R-loops play the primer role in DNA synthesis initiation.
The study found out that the replication of DNA is initiated at CSB and CSBIII regions. Therefore, the transitions of RNA and DNA that are mapped in the study correlate with those of previously done studies. Also, mutations of RNase HI result in increased DNA levels due to the unregulated 7S DNA synthesis. Since RNase HI restricts 7S DNA initiation and mitochondrion DNA to OriH, it eliminates all RNA that has been hybridized to the DNA template except those in the CSB region, to which RNase H1 is the applied as a primer.
The significance of the study is to establish a mechanism for the formation of primer and DNA initiation. R-loop formation and primer processing is crucial in comprehending the functioning of the human mitochondrion
Factors essential for primer formation and replication are influenced by POLγ, a replicative DNA polymerase in the mitochondrion. The replication of mitochondrion DNA is initiated from OriH and OriL. However, the process begins at OriH and results in the formation of H-strands. Following the synthesis of approximately two-thirds of the genome, it then proceeds to the OriL, which becomes available for the RNA polymerase when single-stranded. The process results in the initiation of RNA primers, which are then used to initiate the DNA synthesis by the replicative DNA polymerase, POLγ.
Primers found in the ends of the H-strands are supposed to be removed by nucleases and replaced with DNA. Their removal stems from the fact that nucleases implicated in primer removal have mitochondrion isoforms, including RNase H1, DNA2, and FEN1 (Al-Behadili et al., 2018). When two of these isoforms mutate, the consequences are defects on the mitochondrion DNA and human illnesses. Besides, the mitochondrion has specific nucleases of the DNA known as MGMEI. The nucleases lack isoform and are also incapable of processing RNA flaps. After the removal of the primer, the nicks are ligated by DNA ligase III. Failure to eliminate the from the mitochondrion DNA results in the impairment of ligation and consequently DNA synthesis hence gaps and nicks. The replication cycles can result in double strands at the mitochondrion’s DNA, breaks, and deletions. This makes RNase H1 essential for the primers' processing as it ensures they are not retained at the OriL and OriH. The study aims to address the mechanisms of removing primers at OriL and the crucial role of RNase H1 in this process. However, besides RNase H1, a second nuclease is needed to complete primer removal and ligation at the DNA.
Findings from the study indicate that mitochondrion DNA molecules have long ribonucleotides during replication. This suggests the involvement of RNase H1 in the removal of primer. However, RNase H1 alone is not sufficient as extra nuclease activity is essential for creating ligatable ends to avoid the nicks mentioned above and gaps. Furthermore, demonstrations from the study indicate that during the processing of primers at OriL, RNase H1 leaves unprocessed ribonucleotides at the DNA strands, which sequentially block ligation of DNA (Al-Behadili et al., 2018). The ability of enzyme FENI to cleave short RNA flaps makes it ideal for removing RNA residues left behind by RNase H1.
The study is crucial in establishing that RNase H1 is not completely capable of removing primers at OriL and OriH on its own. Therefore, a nuclease assay such as FENI is crucial for prier processing in vivo. The authors of the study suggest that future research be based on genetic materials to dissect in vivo the mitochondrion and determine whether its nuclease extracts could play the role of FENI.
As aforementioned, RNase H1 is incapable of fully processing the primer. RNase H1 removes RNA strand of DNA and leaves behind two ribonucleotides hanging at the 5’ end of the DNA on a molecular level. Regardless of removing most of the RNA primer, the residues’ removal is crucial for the completion of replication. Nucleases are used for the complete removal of the ribonucleotides. Though less studied, Exonuclease G, abbreviated as EXOG, also removes the residual primers during replication. Its ability to give a FEN 1-like activity stems from its characteristics and capabilities. EXOG, is dimeric and localizes to show 5’-3’ exonuclease and endonuclease activities. Also, it has a sequence homology just as the double ββα -Me nucleases that have sugar nucleases. EXOG also interacts with mitochondrial DNA, mtDNA polymerase POLγ, and DNA Ligase III. As a result of these interactions and characteristics, EXOG can repair single-strand breaks and depletions in the mitochondrial genome. The study aims to demonstrate in vitro characterization, how EXOG can degrade DNA and RNA into dinucleotides.
The study used several substrates to characterize the activity of EXOG in vitro. The first test employed ssDNA substrate to test on single strands of DNA and RNA. Findings indicate that dinucleotides were yielded, and 3 to 7 long products were observed using slightly concentrated EXOG. The degradation nature of EXOG reveals its ability to act as a nuclease for RNA and DNA replication. The next substrate used contained short 5’ DNA flaps. When EXOG was used, the degradation products became 22nt long, revealing its ability to cleave nucleotides from 5’ RNA and DNA strands. The last test was the ability of EXOG to function downstream of RNase H1 at OriL. Results indicate that although EXOG was effective in removing nucleotides, it was more efficient when combined with RNase H1. In vivo, BioID assay proteins interacted with EXOG, resulting in the exonuclease functions.
The study is essential in establishing a nuclease besides FEN1, which can complete the DNA replication cycle. The data and experiments performed indicate that EXOG is a crucial oligonucleotide pathway in DNA mitochondrial essential for replication and maintenance processes.
In conclusion, this paper summarizes the three articles whose studies are centered on human mitochondrion and includes each study's aim, significant findings, and importance. Based on the summary of the three, it is apparent that RNase H1 is essential for primer removal. However, it is not sufficient for the complete elimination of ribonucleotides, hence the necessity for nucleases such as FEN1 and EXOG.
Al-Behadili, A., Uhler, J. P., Berglund, A. K., Peter, B., Doimo, M., Reyes, A., ... & Falkenberg, M. (2018). A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL. Nucleic acids research , 46 (18), 9471-9483. https://doi.org/10.1093/nar/gky708
Posse, V., Al-Behadili, A., Uhler, J. P., Clausen, A. R., Reyes, A., Zeviani, M., ... & Gustafsson, C. M. (2019). RNase H1 directs origin-specific initiation of DNA replication in human mitochondria. PLoS genetics , 15 (1), e1007781. https://doi.org/10.1371/journal.pgen.1007781