DNA repair is a major defense against the second cause of aging described in my treatise Cell DNA Damage. Such repair is absolutely necessary. Damage can be caused by oxidative processes, radiation exposure, and exposure to environmental toxins, cigarette smoke and some antibiotics, and anti-inflammatory drugs(ref). Even without extraordinary exposure, in the course of a normal good day a person may have a million or more events of DNA damage occur in his or her body. Further, the kinds of DNA damage can be of multiple types(ref). Failure to repair damage can lead to cell death, cancer, a number of diseases and premature aging.
In response to this challenge, cells have evolved numerous repair strategies. Some are very clever and still being discovered. I discussed one such line of defense against an important form of DNA damage, double-strand breaks, in my March 2010 blog entry DNA repair cleanup failure – a root cause for cancers? I concluded that entry by saying “There is a lot more interesting research related to DNA repair beyond the thread covered here and I will probably come back to that topic again before too long.” That time is now. I review several additional natural DNA repair strategies together with news of recent discoveries.
Ku and making ends meet
In the earlier blog entry and with respect to the substance Ku that I am concerned with here, the focus is on one particular important kind of breaks, double-strand breaks (DSBs), breaks that can occur naturally in cell differentiation or that are created by radiation and certain chemicals. A double-strand break results in a broken chromosome, and this kind of DNA damage is particularly difficult to repair. Because these breaks completely threaten genomic integrity, evolution has provided us with a number of sophisticated approaches for automatic DNA repair. Non-homologous DNA end-joining (NHEJ) is the main pathway for repairing double-stranded DNA breaks. It functions throughout the cell cycle to repair such lesions. “NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, hallmarks of tumor cells(ref). ”
A protein called Ku has been known for some time to be involved in NHEJ(ref)(ref). A colorful animation of the role of Ku in NHEJ can be found here. Previously, it was thought that Ku worked simply by recognizing broken ends and then recruiting other factors that cleaned up the ends and then joining them again. The ends-cleanup processing is necessary because the strand breaks are often associated with nucleotide damage so that simply connecting ends would result in mutated chromosomes. The April 2010 online publication Ku is a 5â€²-dRP/AP lyase that excises nucleotide damage near broken ends says that Ku does the ends-cleanup job itself. “Ku had previously been presumed only to recognize ends and recruit other factors that process ends; our data support an unexpected direct role for Ku in end-processing steps as well.” As reported in Science Daily quoting Dale Ramsden one of the authors and investigators, “Ku is a very exciting protein because it employs a unique mechanism to repair a particularly drastic form of DNA damage. — Damage to DNA in the form of a broken chromosome, or double strand break, can be very difficult to repair — it is not a clean break and areas along the strand may be damaged at the level of the fundamental building blocks of DNA — called nucleotides — It has been assumed in the past that double strand breaks are the most difficult class of DNA damage to repair and it is often presumed that they simply cannot be repaired accurately.” Part of the importance of this new research is showing that repairs of double-strand breaks can be more accurate than previously thought. Ku-based healing is not only at the chromosome level but also at the nucleotide level.
DNA unwrapping/wrapping in repairing single-strand breaks.
My power boat ties up to the dock using heavy 3-strand polyvinyl rope. If a single strand breaks I can fuse it together with heat from a small blowtorch. But first I must unwrap the strands some to identify the break and make room for the repair. It turns out that the DNA repair machinery does something very similar. The 2009 publication DNA wrapping is required for DNA damage recognition in the Escherichia coli DNA nucleotide excision repair pathway reports on such unwrapping. As explained simply in a Science Daily article: “They found that the proteins that initially recognize the damage amplify the distortion of the DNA around the damaged site by bending the DNA and separating the strands of the double helix. This makes it easier for the next protein to recognize and cut out the damaged portion of the DNA. The cells then patch up the empty space using the healthy half of the DNA as a model to repair the cell to its original state. — The study was conducted using a DNA repair system operated in E. coli, but the findings are applicable to other cells because they adopt similar systems.” The following item relates to the repair process that takes place after the DNA strands unwind.
A shuttlebus first-responder repair protein, SSB
For some time, the protein SSB has been known to play a role in excision repair(ref) of DNA single-strand breaks. The 2009 publication SSB protein diffusion on single-stranded DNA stimulates RecA filament formation lends insight into the ways in which SSB works. “Single-stranded DNA generated in the cell during DNA metabolism is stabilized and protected by binding of ssDNA-binding (SSB) proteins. Escherichia coli SSB, a representative homotetrameric SSB, binds to ssDNA by wrapping the DNA using its four subunits. However, such a tightly wrapped, high-affinity protein–DNA complex still needs to be removed or repositioned quickly for unhindered action of other proteins. — tetrameric SSB can spontaneously migrate along ssDNA. Diffusional migration of SSB helps in the local displacement of SSB by an elongating RecA filament. SSB diffusion also melts short DNA hairpins transiently and stimulates RecA filament elongation on DNA with secondary structure. This observation of diffusional movement of a protein on ssDNA introduces a new model for how an SSB protein can be redistributed, while remaining tightly bound to ssDNA during recombination and repair processes.”
A press release from the University of Illinois explains the actions of SSB in simpler terms. “– a single-stranded DNA-binding protein (SSB), once thought to be a static player among the many molecules that interact with DNA, actually moves back and forth along single-stranded DNA, gradually allowing other proteins to repair, recombine or replicate the strands.” SSB is a first responder. Think of it as a crew first sent out on a small railway shuttlebus car when there is trouble with the tracks. The crew includes the supervisor who will oversee emergency measures and the repairs. “Whenever the double helix of DNA unravels, exposing each strand to the harsh environment of the cell, SSB is usually first on the scene, said University of Illinois physics professor and Howard Hughes Medical Institute investigator Taekjip Ha, who led the study. — Although DNA unwinding is necessary for replication or recombination, it is normally a transient process, he said. Exposed single-stranded DNA (ssDNA) can be damaged or degraded by enzymes in the cell. Damaged DNA may also come unwound, and ssDNA can bond to itself, forming hairpin loops and other problematic structures. — “If you have lots of single-stranded DNA in the cell, basically it’s a sign of trouble,” Ha said. “SSB needs to come and bind to it to protect it from degradation and to control what kind of proteins have access to the single-stranded DNA.” Although other proteins are known to travel along double-stranded DNA, this is the first study to find a protein that migrates back and forth randomly on single-stranded DNA, Ha said.”
“– the researchers showed that SSB diffuses randomly back and forth along single-stranded DNA, and that this movement is independent of the sequence of nucleotides that make up the DNA. They also found that an important DNA repair protein in E. coli, RecA, grows along the ssDNA in tandem with the movement of SSB. As the RecA protein extends along the DNA strand it prevents the backward movement of the SSB. — The researchers also found that SSB can “melt” small hairpin loops that appear in single-stranded DNA, straightening them so that the RecA protein can bind to and repair them. In this way SSB modulates the activity of RecA and other proteins that are involved in DNA repair, recombination and replication. — “SSB may be a master coordinator of all these important processes,” Ha said(ref).”
The role of HMGB1
No, HMGB1 is not an agency in the Russian secret service. HMGB1 stands for high mobility group box protein 1. It is a protein that pays an important role in DNA repair, though what to do about it is controversial. HMGB1 is an Alarmin. “Alarmins are a newly described and still emerging group of structurally diverse, but functionally related, molecules that include defensins, cathelicidins, eosinophil-derived neurotoxin, and HMGB1 — All are released in response to infection and tissue damage, and mediate innate immunity and tissue repair(ref).” Let’s start with the bad reputation for HMGB1, which is that it causes inflammation and plays a role in creating epileptic episodes and is implicit in the progress of many cancers. For example the medical news report Salute: epilessia? Ã¨ colpa della molecola HMGB1 translate into Health: epilepsy? It the fault of the molecule HMGB1. Many publications suggest that targeting HMGB1 could provide effective cancer therapies(ref). The 2003 publication Dealing with death: HMGB1 as a novel target for cancer therapy suggests the development of anti-cancer drugs that work by inhibiting HMG1. HMGB1 is thought to play a key role in chronic inflammatory autoimmune disease and as well as in severe, acute systemic inflammatory disease(ref). ”Because HMGB1 plays a key role in inflammation, it’s also being targeted in drugs under development for rheumatoid arthritis and sepsis. “ A quite different view of HMGB1 is suggested in the 2008 medical news report Suspect protein HMGB1 found to promote DNA repair, prevent cancer. “An abundant chromosomal protein that binds to damaged DNA prevents cancer development by enhancing DNA repair”– “Long known to attach to sites of damaged DNA, the protein was suspected of preventing repair. “That did not make sense to us, because HMGB1 is a chromosomal protein that’s so abundant that it would be hard to imagine cell repair happening at all if that were the case,” Vasquez (senior researcher in the study) said. In a series of experiments — Vasquez and first author Sabine Lange, — tracked the protein’s impact on all three steps of DNA restoration: access to damage, repair and repackaging of the original structure, a combination of DNA and histone proteins called chromatin. — First, they knocked out the gene mouse embryonic cells and then exposed cells to two types of DNA-damaging agents. One was UV light, the other a chemotherapy called psoralen that’s activated by exposure to darker, low frequency light known as UVA. In both cases, the cells survived at a steeply lower rate after DNA damage than did normal cells. — Next they exposed HMGB1 knockout cells and normal cells to psoralen and assessed the rate of genetic mutation. The knockout cells had a mutation frequency more than double that of normal cells, however, there was no effect on the types of mutation that occurred. –Knock out and normal cells were then exposed to UV light and suffered the same amount of damage. However, those with HMGB1 had two to three times the repair as those without. Evidence suggests that HMGB1 works by summoning other DNA repair factors to the damaged site, Vasquez said.”
Going back to the issue discussed above of DNA unwrapping/wrapping in repairing single-strand breaks, HMGB1 apparently plays an important role in that process. “Lange and Vasquez hypothesize that HMGB1 normally binds to the entrance and exit of DNA nucleosomes, so is nearby when DNA damage occurs. It then binds to and bends the damaged site at a 90-degree angle, a distortion that may help DNA repair factors recognize and repair the damage. After repair it facilitates restructuring of the chromatin(ref).”
“Pinpointing HMGB1’s role in repair raises a fundamental question about drugs under development to block the protein, Vasquez said.” “Our findings suggest that depleting this protein may leave patients more vulnerable to developing cancer.” I wonder if this view is giving any pause to those who are out pushing the development of HMGB1 inhibitors as drug candidates? I think it should.
I expect I will be coming back to DNA repair yet-again before too long.