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Tag: Recombination

Somatic Recombination Part 4: Cutting up your DNA

by on Mar.06, 2011, under Immunology

So you’ve been told that all the cells in your body contain the same genetic material? I’ve seen it written as a general principle of Genetics and a quick search around the web will yield many sources indicating that all somatic cells contain the exact same DNA. I did find a few articles in science magazines which “revealed” that this may not be the case, but the general consensus that they do was prevalent.

From all I’ve read, research and texts included, the DNA contained in all somatic cells is the same with one exception; the hematopoietic cells. These cells are the cells of the blood and they all descend from a single progenitor, the Human Hematopoietic Stem cells which can be found in bone marrow. These stem cells give rise to a diverse population of progeny including erythrocytes (red blood cells), T Cells, B Cells, Macrophages, and Natural Killer cells. To be accurate, the stem cells and their immediate descendants contain “germ-line” DNA, which is DNA that was formed during the individual’s fertilization event and is shared with virtually every other somatic cell in the body. However, as the hematopoietic descendants start to specify into the progenitors of T and B cells or “lymphopoietic” progenitors, the process of Somatic Recombination which creates the antigen receptors I’ve been writing about cuts out and discards parts of certain chromosomes thus creating genetically distinct human cells within our own bodies.

In my last post I presented the gene loci on which this process acts and highlighted the first actors in the process; the Recombination Activating Gene 1 & 2 proteins (RAG1 & RAG2). The arrangement of the gene loci is very important in this discussion and the rest of what I’m about to present doesn’t make much sense without this knowledge, so you can click this link to read that post if necessary. The process is performed by the V(D)J Recombinase and the process progresses as the molecules that make it up are assembled, I will introduce them as I describe the process.

It begins with two molecules each containing one RAG1 and one RAG2. These complexes then randomly bind, one each, to two of the hundreds of Recombination Signaling Sequences (RSSs) found surrounding each gene segment in the target antigen locus. It should be noted that the selection is not random across the segment. For example, two V regions will not join; it must be a V and J, for example, in a light chain locus. After binding, the two complexes come together and bind resulting in the formation of a loop of DNA between them which contains all the gene segments that may have been between the two segments brought together.

With the loop formed, the RAG complex will cut the strand exactly at the points where the RSS ends and the coding segment begins (see diagram below). This happens because the RAG complex has an inherent ability to act as an endonuclease. This essentially means that it is a protein that can break a DNA molecule into parts. This is the action that excises a piece of our DNA which is called the signal joint (because the RSS of each segment are joined flush). At this point, because of reasons relating to the chemical properties of the structure of a molecule of DNA, the ends of each strand which are cut fuse to form hairpins.

At this point other proteins join the complex to complete the recombinase and the process. These proteins are actually ubiquitous in human cells and I believe they are present for every mitotic and meiotic event that occurs over an individual’s lifetime. The first two proteins to bind to the growing V(D)J Recombinase are named Ku70:80 and they hold together both the ends of the chromosomal DNA strand, and those of the signal joint. Note, that this is the last I will mention the signal joint as once it’s ends are fused, it is no longer part of our story.

It is here where the process becomes a little unnerving for me. The very precise cut made by the RAG1/RAG2 complex to setup the joining of gene segments is one thing, but what happens next, to me, is another. The following video presents an excellent visualization of the entire process with a voice-over by Dr. Julie Theriot; a brilliant mind. I will, however, go into a little more detail below.

A protein named DNA-PK, or DNA-dependent Protein Kinase, is formed when another protein named DNA-PKcs binds tightly with Ku after the strands are bound. And to this another protein named Artemis joins. Artemis is also an endonuclease without which we would be ending our story with a broken strand of DNA. Artemis cuts the hair pins formed after RAG1/RAG2 excised the signal joint, but the location of its cut appears to be random. It has been found that they occur anywhere along the hair pin and this is where the fun starts.

With the location being random, unequal ends are often left which must be equalized before the DNA can be rejoined. Enter another protein that is found in all our cells and has a great name; Terminal Deoxynucleotidyl Transferase (TdT). TdT randomly adds bases to the cut ends resulting in what is essentially a newly formed random sequence on each. At the same time, two more newcomers bind to each other and then the Recombinase; DNA Ligase IV and XCR44. There are ubiquitous DNA repair proteins which and as soon as they bind, they start trying to re-join the strands; the name Ligase actually comes from the Latin “ligare” meaning “to bind or tie”. With TdT inserting random bases, the repair/ligation complex may need to knock out and replace non-complementary pairings such as a Guanine to a Thiamine.

With the ligation effort winning out in the end and the chromatid now back in one piece, the process of Somatic Recombination is complete. With just a few more steps we have a complete gene which, when transcribed will form one of the two chains in an Immunoglobulin. The process, however, has left the chromatid irrevocably changed; it is unique, different from any other member of its chromosome pair in the body, including other B cells. I’d like to note again that this change is key to the efficacy of our adaptive immune response. Without the enormous diversity generated by this process, neither the B and T cell antigen receptors nor the antibodies secreted by the former would be capable of binding effectively to the diverse and ever changing peptides making up evolving pathogens. However, it also introduces a potential for a number of pathologies, including cancer.

It has been shown in research1 that if the DNA repair mechanisms like DNA Ligase IV\XCR44 and the DNA damage checkpoints are not functioning properly, Somatic Recombination can result in several pathologies. These include aneuploidy, an abnormal number of chromosomes, translocations, where one part of a chromosome is fused with another, and neoplastic transformation, cancer.

In fact, according to both Janeway’s Immuno Biology (p 312) and a paper published in Nature Immunology in 20071, translocations are found in most lymphoma tissues. What is compelling for me about these translocations is that they most often involve what are known as proto-oncogenes which are genes encoding proteins that, when not functioning properly, cause cancer.

What causes DNA checkpoints and the DNA repair proteins to malfunction? I’ve seen the research showing the obvious conclusion; mutations forming in the genes coding for the proteins which carry out the functions. However, I suspect epigenetics may play a role in at least some of the scenarios. What causes these translocations? Given the fact that many lymphomas are age onset, I would conclude that probability must be considered, but the translocations of proto-oncogenes suggest to me that epigenetic factors should be examined. There are genes such as Myc (Myelocytomatosis)that regulate the expression of up to 15% of our entire genome and do so through epigenetic factors such as HAT (Histone Acetyltransferase). It should not be surprising that Myc is an oncogene and it is, in fact, one that is involved in the translocations found in lymphoma tissues.

1. Nature Immunology 8, 801 – 808 (2007)
Published online: 19 July 2007 | doi:10.1038/

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Somatic Recombination, Part 1: Immunology; saving and speaking of life.

by on Dec.29, 2010, under Immunology

I would like to share with you some knowledge that very few people in the world have.  The topic is a very hearty one; Immunology, considered to be one of the most difficult in the health sciences.  However, the immune system is of critical importance not only in defeating pathogens such as bacteria and viruses, but also in clearing the body of damaged tissue and eliminating cancerous growths.  I will introduce some vocabulary for the simple reason that without it, this post would become very long, even for me.  It is said that a picture is worth a thousand words, well, in Immunology, some words are worth about the same.  The source of the information and my personal opinions presented in this post is my study of Janeway’s Immuno Biology.  I’m not a medical professional, and nothing I present in this post, or any other post I may make on a health science topic, should in any way be considered medical advice, the intent of the authors of any of the texts I reference, or an authoritative explanation.  This post is solely my understanding, theories, and interpretation of the material I’ve studied.

In a short series of posts, I will introduce an immune system process with the impressive title; Somatic Recombination, or, in this context,  V(D)J Recombination.  This process is significant for many reasons outside its critical and sole function; to build receptors.  These receptors, also called antibodies, or Immunoglobulins, are capable of recognizing bits and pieces of invading pathogens and other substances that might harm the body.  Of particular significance, the building process has potentially dangerous ends because it includes not only the cutting out and elimination of sections of an individuals DNA, but also the intentional introduction of mutations into the recombined DNA strands.  Also, the process, when viewed across many Immune System cells, is an example of selection.  It is not the same, of course, as natural selection occurring at a macroscopic level, but at the basic level, it does as it involves the survival and proliferation of those cells with receptors containing the sequence of amino acids that will best bind to a given antigen; the name given to the bits and pieces which stimulate our immune system into action.

Another interesting note about this process is that it is closely related to the process by which retroviruses insert their genetic information into the DNA of their host cells, i.e. human cells, which in turn can cause disease.  In fact, the RAG (Recombination-Activating Gene) protein which directs the process is arranged differently from other human genes suggesting that it was evolutionarily adapted into our genome making Somatic Recombination possible. 

I have often felt, during my studies that humans seem to be fabric into which is woven parts of many different molecules and organisms that probably coexisted in mutually beneficial societies; a social symbiosis.  I am of the opinion that this symbiosis is the key to the successful evolution of life rather than the necessary, but misinterpreted “survival of the fittest” notion to which evolution is so often tied.  Questioning Darwin… talk about delusions of grandeur on my part.

The Immune System, so far in my study, looks to me like a living model through which the evolution of life on Earth can be seen.  First, it was necessary for me to resist the compulsion to anthropomorphize.  The Macrophage, a staple cell of the Immune System, was so named because it was seen as a “big eater”.  I think this view is detrimental to a clear understanding and research path because we are essentially speaking of energetically favorable reactions not cells with a hankering for vittles.  When I remove this lens, I see the symbiotic relationship with mitochondria as possibly allowing the re-tasking of complex energy seeking molecules to protective capacities and the establishment of self at the cellular level.  I see that its not a matter of destroying non-self, there is actually more non-self inside us than self, it’s a matter of information dissemination, examination, and a resultant action.  To illustrate, consider that molecules called MHC which present pathogen derived peptides to T cells and elicit an immuno-protective response.  These MHC molecules, will present peptides from the individual’s own healthy proteins in the same manor if an infection is not present.  This presentation should, it would seem,  result in no action by the T cells, but considering auto-immune disorders of unknown pathogenesis, perhaps this is not always the case.  Pathogenesis is the origination and development of a disease.

This is just the very tip of a large and fascinating iceberg.  I hope my explanation of Somatic Recombination in antibody production serves well in sparking interest in a study which has saved countless lives and may yet shed light on the evolution of life  itself.

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