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DNA Photolyase

  

Introduction

 

DNA photolyase is a protein that repairs direct chromosomal damage caused by UV light, typically UVB radiation in the blue-light range (290-320 nm), through a process called photoreactivation (Masson F. et al., 2009). There are two main photolyases that differ in the types of damage they repair: cyclobutane pyrimidine dimmers (CPDs) are repaired by CPD photolyase and have been found in organisms from all three kingdoms, 6-4 photoproducts (6-4 PPs) are less frequent and are repaired by 6-4 photolyases (Lucas-Liedo et al, 2009). While CPD and 6-4 PP lesions occur in all organisms exposed to high levels of UV light, not all organisms contain the photolyase protein, and therefore, they can not directly repair the damage. All placental animals, including humans, seem to lack photolyase activity, and thus they must rely on nucleotide excision repair to correct the lesions (Masson F. et al., 2009). 

 

 

Structure of DNA Photolyase

 

DNA photolyases typically consist of between 454 and 614 amino acids, and they are made up of two structural domains, an alpha/beta domain and a helical domain, which are attached by a linker region (Harrison, C.B. et al., 2005, Lucas-Liedo et al, 2009). Two chromophore cofactors bind to the phyotolyase to activate it (Lucas-Liedo et al, 2009). The first cofactor binds to the alpha/beta region of the photolyase and can vary from organism to organism (Harrison, C.B. et al., 2005). This cofactor is responsible for acting as a light-harvesting antenna (Harrison, C.B. et al., 2005). The second cofactor is FADH in all organisms, and it is a catalytic chromophore that binds to the helical domain of the photolyase (Lucas-Liedo et al, 2009). 

 

CPD Photolyase                                                                                                   6-4 PP Photolyase

 

 

 

 

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Photolyase Activity 

 

 

Types of Damage Repaired by Photolyase

 

Exposure to UV radiation can cause adjacent pyrimidine nucleotides, thymine or cytosine, in a cell's DNA to covalently bind to one another and form a dimer. CPDs are formed when the carbon atoms C-5 and C-6 covalently bind to those same carbon atoms in an adjacent pyrimidine, and 6-4 PPs are formed when a C-6 atom from one pyrimidine covalently binds to a C-4 atom in an adjacent molecule (Tremblay, M. et al., 2009). The picture below shows how adjacent thymines can be altered by UV radiation to form the CPD or 6-4 PP lesions.  

 

 

The dimers cause a helical distortion that affects proper base pairing of the nucleotides and leads to disruptions of the transcription and replication processes (Tremblay, M. et al., 2009, Schul, W. et al., 2002). Depending on the DNA sequence around the dimer, both dimer forms can disrupt the stability of the DNA's nucleosomes to varying degrees (Tremblay, M. et al., 2009). Disruptions also occur because the improper DNA bending can change the way that proteins interact with the DNA (Tremblay, M. et al., 2009). A high degree of disruption can cause the cell's cycle to stop, induce apoptosis of the cell, or lead to cancer by causing the cell to bypass the mechanisms that control growth (Tremblay, M. et al., 2009, Schul, W. et al., 2002). 

 

CPD lesions account for 70-80% of the UV damage observed while 6-4 PP lesions account for 20-30% of the damage (Tremblay, M. et al., 2009). In addition to the CPDs being more frequent, they are also more efficiently corrected by the cell's repair mechanisms (Young-Hyun You et al., 2001). Therefore, studies suggest that 6-4 PPs may be more mutagenic (Young-Hyun You et al., 2001). This may also be due to the inability of the cell to correctly bypass the 6-4 PPs during replication and transcription (Young-Hyun You et al., 2001). Specifically, DNA polymerase mu has been shown to play an important role in bypassing unrepaired damage (Young-Hyun You et al., 2001).

 

 

How Does Photolyase Recognize the DNA Damage?

 

The exact mechanism of how the photolyase is able to detect the dimers in the DNA molecule is still unknown (Tremblay, M. et al., 2009). However, it is believed that the phosphates in the DNA backbone near the dimer play a key role in the photolyase's ability to identify and repair the lesion (Masson F. et al., 2009). The phosphates and photolyase interact with one another by forming salt bridges and hydrogen bonds that help to stabilize the photolyase-DNA complex so that the dimer can be removed (Masson F. et al., 2009). Also, histone modification and chromatin remodeling can greatly affect photolyase's ability to recognize the damaged areas since the presence of nucleosomes seems to restrict photolyase activity by making the DNA wrapped around them less accessible to the protein (Tremblay, M. et al., 2009).

 

 

How Does Photolyase Repair the Damaged Area?

 

Photolyase repairs the damaged DNA through a process called photoreactivation. The CPD photolyase activity is composed of two main reactions, a light-independent reaction and a light-dependent reaction. During the light-independent reaction, the bound photolyase causes the CPD to flip into the protein's active site (Tremblay, M. et al., 2009). The actual functioning of the protein is light-dependent, and it requires the light-harnessing cofactor to absorb light energy (350-450 nm) (Tremblay, M. et al., 2009). The other catalytic cofactor is in a reduced state so that when the absorbed light energy is transfered to it, the FADH- becomes excited and donates an electron to the CPD. The addition of this electron to the CPD disrupts the covalent bonds between the C-5 atoms and C-6 atoms so that the bonds break and the pyrimidine molecules are returned to their undamaged states (Masson F. et al., 2009). Once the damage is repaired, the electron is transfered back to the FADH cofactor so that it returns to its reduced state and therefore regains its catalytic activity (Masson F. et al., 2009). The photolyase has less affinity for the repaired DNA, and is thus able to dissociate from the DNA once the repair is made (Maul, M.J. et al., 2008).

 

The above image shows how an electron is transfered from the excited FADH to the dimer (left), the electron

then disrupts the carbon bonds in the dimer (center), and the electron is returned to the FADH (right)

 

 

The repair mechanism of 6-4 PP photolyase is less studied than that of CPD photolyase, and therefore, the mechanism is not fully understood (Maul, M.J. et al., 2008). The basic mechanisms of 6-4 PP's repair are very similar to the repair of CPDs. However, for a detailed description of the process refer to the study by Maul M.J. et al., 2008.

 

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Evolutionary History of Photolyase

 

 

When did Photolyase Evolve Compared to Other DNA Repair Processes?

 

The fact that photoreactivation activity exists over a wide taxonomic range and was present in the last common ancestor of all cellular life forms, is evidence that the origin of photolyase occurred very early on in the evolutionary process (Lucas-Liedo et al, 2009). It is believed that photolyase activity predates the origin of eukaryotes and the atmospheric ozone layer, and therefore, it was one of the very first repair mechanisms to develop (Lucas-Liedo et al, 2009). 

 

As organisms continued to evolve and become more complex, more complex repair mechanisms were also developed. Presently, there exist many repair pathways that are highly redundant and can act as backups for one another (O'Brien, P.J., 2006). Thus, nucleotide excision repair (NER) is a much more complex repair process that has evolved and can remove the same types of DNA damage as photolyase. For more information on the mechanism of NER visit these websites: (NER in Eukaryotes & NER Overview). 

 

 

 

What Species Have Lost Photolyase Activity and Why?

 

It is apparent that DNA damage can lead to aging, apoptosis, and cancer, and therefore, it is beneficial for organisms to be able to correct damaged areas. Yet, despite the advantage of possessing photolyase activity, many species have lost the protein throughout evolutionary history (Lucas-Liedo et al, 2009). All placental animals, including humans, have lost photolyase activity, and instead, they must rely on NER to remove the CPDs and 6-4 PPs (Masson F. et al., 2009). The NER process is much more complex than the photoreactivation process, and it has been shown to be less efficient at repairing lesions caused by UV radiation (Lucas-Liedo et al, 2009). NER is especially slow at removing CPDs, which typically take more than 24 hours to repair (Tremblay, M. et al., 2009, Schul, W. et al., 2002). This low efficiency causes people with inactivating mutations in the genes involved in NER to be highly sensitive to sun exposure since they are completely dependent on this mechanism to correct UV-induced DNA damage, and defects in the NER pathway cause disorders such as Xeroderma Pigmentosum and Cockayne Syndrome (Lucas-Liedo et al, 2009, Tremblay, M. et al., 2009, Schul, W. et al., 2002). For more information on what species have lost photolyase activity and why, refer to the Lucas-Liedo et al, 2009.

 

 

Evolution of Cryptochromes from Photolyase

 

Cryptochromes (CRYs) are blue-light receptor proteins that seem to have evolved from photolyase (Bayram et al., 2008). Although the cryptochromes appear to be very similar in structure to photolyase, they do not appear to have any photolyase activity (Lucas-Liedo et al, 2009). Instead, CRYs regulate the growth, development and circadian rhythm of many organisms (Bayram et al., 2008). For more information on the many functions of CRYs and how they have evolved from photolyases refer to these articles: (Bayram et al., 2008), (CRY magnetic sensing), (ScienceWeek), (Animal CRYs), (Levy et al, 2007).

 

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Future Uses of DNA Photolyase

 

 

Improved UV Resistance in Photolyase Transgenic Mice

 

 Many experiments have been done to determine how placental animals will respond to being induced with the photolyase gene. One study by Schul W. et al. integrated the gene for CPD photolyase from a marsupial, P. tridactylis, into the genome of mice (Tremblay, M. et al., 2009, Schul, W. et al., 2002). They exposed the mice to UV radiation and used immunofluorescent labeling of the CPD and 6-4 PP lesions to determine if the photolyase had been successful in repairing the damage (Tremblay, M. et al., 2009, Schul, W. et al., 2002). The mice that were kept in the dark for one hour after the initial UV exposure, thus preventing the photolyase from receiving the necessary light energy needed for enzymatic activity, still contained both CPD and 6-4 PP lesions in their DNA (Tremblay, M. et al., 2009, Schul, W. et al., 2002). This result indicated that NER provides a very low level of repair in that first hour (Tremblay, M. et al., 2009, Schul, W. et al., 2002). However, the mice that were exposed to photoractivating light after the initial UV exposure had CPD lesions that were hardly detectable, and this result showed that the CPD photolyase gene was highly successful in repairing the damage (Tremblay, M. et al., 2009, Schul, W. et al., 2002).

 

 

This image shows the results of the Schul W et al study. The lighted areas are the result of the immunofluorescent labeling of the CPD and 6-4 PP lesions, and therefore they represent the cell nuclei that contain the UV-induced lesions.

 

 

The study further showed that quick removal of the CPDs made the skin cells significantly less sensitive to the UV light exposure (Tremblay, M. et al., 2009, Schul, W. et al., 2002). A larger number of the photolyase-containing cells survived the exposure as compared to the cells that did not contain the photolyase gene or did not receive the photoractivating light after the initial UV exposure (Tremblay, M. et al., 2009, Schul, W. et al., 2002). The CPD photolyase had a 2-fold dose reducing effect on the level of UV radiation that the cells were exposed to (Tremblay, M. et al., 2009, Schul, W. et al., 2002). This means that the number of cells that survived a UV exposure of a particular dose corresponds to the number of cells that typically survive at half of that UV exposure (Tremblay, M. et al., 2009, Schul, W. et al., 2002). 

 

The study also examined the effect of CPD photolyase on the induction of apoptosis (Tremblay, M. et al., 2009, Schul, W. et al., 2002). To examine this, a section of hair was removed from the dorsal side of the mice and the exposed skin received a dose of UV radiation. The mice that were subsequently exposed to 3 hours of photoreactivating light showed little to no signs of apoptosis, while the mice that were not exposed to subsequent photoreactivating light showed high levels of apoptosis (Tremblay, M. et al., 2009, Schul, W. et al., 2002).

 

Human cell lines have also been developed that contain a transferred marsupial photolyase gene (Chigancas, V. et al., 2000). Like the mice cells, the human cells are also able to partially remove CPDs, and the photolyase increases the cells' resistance to the damaging effects of UV radiation (Chigancas, V. et al., 2000). 

 

 

Topical Use of Photolyase

 

The recent research on the benefits of DNA photolyase in repairing UV-induced skin damage has led to the development of a CPD photolyase-containing liposome that can be applied topically to UV-exposed skin (Tremblay, M. et al., 2009, Schul, W. et al., 2002, Jans, J. et al., 2005). It was shown that dimer repair can be improved by 45% when this lotion is applied (Masson F. et al., 2009). While topical application will only result in less than half of the dimers being repaired, the application could also help to prevent sunburn cell formation (Tremblay, M. et al., 2009, Schul, W. et al., 2002). However, the use of this lotion requires a very careful and timely application in order to be effective, and it is also only active for a short period of time (Jans, J. et al., 2005). With further research these photolyase-containing products will hopefully be able to reduce the number of individuals who develop various forms of skin cancer and may offer great hope for individuals suffering from disorders that make them extremely sensitive to UV light. The following video shows a women who is receiving an experimental treatment of topically applied photolyase.

 

 

 

 

 

 

 

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