Structural Biochemistry/Maintaining Genome Integrity

In the article, "The interface between transcription and mechanisms maintaining genome integrity," the main interfaces between transcription and other process related to DNA and the relevance to genome integrity in eukaryotes is thoroughly explored. Researchers at Cancer Research UK London Research Institute have studied the process of transcription, translation, and RNA mutagenesis and its effect on maintaining genome integrity. Specifically, eukaryotic RNA polymerase II transcription affects processes like chromatin remodeling and DNA repair. Loss of genome integrity causes changes in gene expression. For example loss in a chromosome region causes a mutation and in turn alters the protein that is created based on the genetic code. The research group has also discovered that the movement of RNA polymerases through the chromatin directly and indirectly affects the integrity of the genomic region that is transcribed.

Obstacles to transcription The factors that affect the maintenance of genome integrity during the transcription process are nucleosomes and DNA damager. RNA polymerase and its co-factors attempt to temporarily move the obstructing nucleosomes away from the transcribing region of the DNA. Second, DNA damage like a DNA double-strand break will not allow the RNA polymerase to continue transcribing since the bulky DNA lesion acts as a large obstacle. Thankfully, simple base damage is not a permanent obstacle as there are developed pathways that attempt to fix the lesion.

The pathway that fix the damage-stalling DNA lesion is called TC-NER, or transcription coupled nucleotide excision machinery. The TC-NER pathway attempts to remove areas of DNA damage in the transcribed region that is first encountered by the RNA polymerase II. If there are lesions further down the transcribed region that the RNA polymerase has not yet reached, another pathway called the GG-NER, or general genome nucleotide excision machinery is used. The TC-NER is dependent on proteins called Cockayne Syndrome (CS) A and CSB, that act as cofactors in this pathway. However, the exact mechanism by which the CS protein help catalyze the removal of DNA lesions through TC-NER is still to be determined.

A third pathway (in addition to TC-NER and GG-NER) used to fix the damage-stalling is RNA Polymerase II polyubiquitylation. The polyubiquitylation process has 3 types. The RNA Polymerase II K63 pathway is independent and does not lead to the degradation of the RNA polymerase. However, in the more direct pathway RNA polymerase II is subject to mono-ubiquitylation and the K46 poly-ubiquitylation. After these two steps, the polymerase is degraded. This degradation of the polymerase itself is typically used as the last result to fixing the damage-stalling DNA with the bulky lesion. Once the polymerase is degraded, the DNA lesion is attempted to be removed through a second trial of TC-NER and GG-NER when another RNA polymerase II encounters the lesion, or by DNA recombination.

Another phenomenon that affects genome integrity occurs during the process of DNA replication. During DNA replication, a replication fork is formed where there is a leading and lagging strand that make up the two sides of the fork. At the center of the fork, DNA polymerase slides along and replicates the DNA template. Pol ε is responsible for the leading strand synthesis and the Pol δ is responsible for lagging strand synthesis. In addition to the polymerases, there are primases, helicases and supplemental enzymes all present at this replication fork. The collision of these individual machineries can evidently cause severe consequences.

The realization that these clashes can occur at the replication fork provide further evidence of the phenomenon of transcription-associated mutagenesis or TAM. Highly transcribed region in the genome tend to have a lower percentage of packaging aids like nucleosome present. This leads to a more open structure and 'single strandedness'. The loss of the chromatin proteins and the resulting structural packaging damage of the DNA strands leave the DNA more susceptible to damage and loss of genome integrity. Overall, the spontaneous mutation rate in a eukaryotic gene is proportional to the transcription level. Interestingly, this means that the movement of RNA polymerase II and the repeating transcription processes of the same segment on the DNA strand of interest leading a higher probability of mutagenesis. An example of such mutation has been determined by the research group. In yeast DNA, there is accumulation of dUTP instead of dTTP during DNA replication as a result of highly transcribing a particular strand. This example leads to the conclusion that replication fork breakdown does occur if there is clashing when transcription is in process as well.

RNA mutagenesis does not contribute significantly to the loss of genome integrity like DNA mutagenesis does. This is because the mRNA that is used to eventually produce proteins are short-lived, especially in comparison to tRNA and mrRNA. Due to the short life of mRNA, it is doubtful of mutant protein is caused by RNA mutagenesis.

In conclusion, during transcription and replication of DNA, a loss of genome integrity may occur as a result to clashes and lesions of the proteins and polymerases in the normal process. The cell uses pathways like TC-NER,GG-NER, and RNA PII ubiquitylation to attempt to remove the lesions. Better computational models for studying these pathways may help understand genome instability thoroughly in the future.

Reference 1. Svejstrup, Jesper Q. The interface between transcription and mechanisms maintaining genome integrity. Clare Hall Laboratories, Cancer Research UK London Research Institute, Blanche Lane, South Mimms, EN6 3LD, UK. Trends in Biochemical Sciences Vol. 35 No. 6

2. Mefford, H.C and Eichler, E.E. (2009). Duplication hotspots, rare genomic disorders, and common disease. Curr, Opin. Genet. Dev. 19, 196-204.