Monthly Archives: November 2016

This is the fourth and final part in a series of explainers on molecular biology topics pertinent to my recently published  PhD thesis work. See part 1 about DNA damage part 2 about damage repair, and part 3 about transcription. Unlike those posts, this last part is a little heavy on the science jargon and is intended for a scientific audience, so if this isn’t your area I suggest you check out parts 1-3!

In part 3 I left off with how RNA can invade DNA, forming a potentially dangerous RNA-DNA hybrid structure. When RNA invades duplex DNA, the resulting three-stranded structure is referred to as an R-loop. These R-loop structures have been found in the cells of many organisms, from bacteria, to yeast, to plants and humans. Cells which accumulate high levels of R-loops have high levels of DNA damage and chromosome instability. However, cells have mechanisms in place to remove and process R-loops to prevent their accumulation. All cells, from bacteria to humans, posses two enzymes that can degrade the RNA in an R-loop called RNase H1 and RNase H2. It is puzzling that cells have two enzymes that perform overlapping functions, but their evolutionary conservation indicates that they both perform indispensable jobs. Both RNase H1 and RNase H2 are each required for viability in mice and human cells, and small mutations in RNase H2 cause a neuroinflammatory autoimmune disease called Aicardi-Goutieres Syndrome in human patients.

Fortunately, RNase H1 and RNase H2 are not required for life in the simpler, single-celled organism of budding yeast, and this allowed me to study their function. I knocked out either RNase H1, H2, or both from the yeast genome and observed the effect on chromosome instability. I confirmed previous reports that cells lacking both RNase H1 and H2 had elevated levels of chromosome instability. However, I also found that the lack of only RNase H2 lead to high levels of chromosome instability, while the lack of only RNase H1 did not have elevated instability.

To add another layer of complexity, RNase H2 not only removes long molecules of RNA that form R-loops, but it also can remove single ribonucleotides that are mistakenly incorporated into the DNA strand during DNA replication. There was some controversy in the field as to which function of RNase H2 is the crucial one to prevent chromosome instability.  I determined that it was specifically the R-loop removal activity of RNase H2, not the single ribonucleotide removal activity, that was important for preventing chromosome instability. Lastly I found evidence that RNase H2 acts on hybrids genome-wide to prevent chromosome instability. In contrast, RNase H1 acts at a subset of hybrids to repress locus-restricted chromosome instability.

As R-loops have emerged as a source of DNA damage and chromosome instability, we would like to understand the systems which remove them from the genome. This study presented evidence that RNase H1 and H2 play similar yet separated roles in protecting the genome. This provided insight into why these enzymes, which have overlapping R-loop-removal activities, have been conserved throughout evolution.

This is the third in a series of explainers on molecular biology topics pertinent to my recently published  PhD thesis work. See part 1 about DNA damage and part 2 about damage repair.

As we saw in parts 1 and 2, cells work really hard to protect their DNA. But what is it all for? How is that precious information used to build cells and make entire humans? You may have heard that genes make us who we are. Well, genes are segments of DNA which instruct the assembly of proteins. Proteins are the real workhorses of the cell, they do everything including the cellular processes I talked about in parts 1 and 2. Proteins duplicate the DNA before cell division, they package up the DNA into chromosomes, and repair DNA. Parts of your body outside of the cell like your hair and the surface of your skin are also made of proteins. Proteins are everywhere doing everything!

DNA holds the information to make proteins, but it is too precious to get directly involved in the nitty gritty business of making them. It stays all safe and snug in its own section of the cell called the nucleus, and the genetic information is copied down into a molecule called RNA, which leaves the nucleus and directs the building of proteins. RNA is a molecule very similar to DNA, but it is much more disposable. Once it has done its job to make a protein, it is broken down and its parts are recycled. This process of copying DNA into RNA is called transcription.

Since transcription is so fundamental to and necessary for life, and it seems to be a process put in place to protect DNA, researchers were surprised to find that transcription itself can actually be a source of DNA damage. Over the past decade we have found that RNA can actually invade DNA forming an unstable structure. This RNA-DNA structure can lead to DNA damage, with all the potentially disastrous consequences.

Transcription is constantly happening as cells are always making new RNA and proteins. But this RNA is potentially damaging to the DNA, so what’s a cell to do? Luckily there are back-up systems in place. I studied two proteins called RNase H1 and RNase H2 which can remove the RNA from DNA and resolve the problem. We still aren’t exactly sure how RNA in the DNA causes damage, but cells that lack functional RNase H1 or RNase H2 have a lot of DNA damage, and humans cannot survive without these enzymes. For my work I studied how these two proteins act in similar and different ways to reduce DNA damage. In part 4 of this series I will go into more details about the study.