Repair mechanisms

Not all exposure to UVR can be avoided and therefore all organisms require some capacity to repair damage caused by these wavelengths. Little is known about the mechanisms that recognize and repair damage induced by UVR in marine and freshwater systems and most of what is known about repair of UVR-induced DNA damage comes from research on bacteria, which has been applied to aquatic organisms. Repair of DNA and of proteins may be induced during exposure to UVR and may continue well after the exposure to damaging wavelengths has ceased.

10.4.1 Protein repair mechanisms

Repair proteins are induced by exposure to UVR in amoebae and cyanobacteria and de novo protein biosynthesis has been shown in a variety of systems. Turnover of damaged proteins is an important mechanism in overcoming damage by UVR. Reactivation of damaged photosystems after prolonged exposure to UV-A was important in the recovery of the diatom Melosira sp. and of the green alga Chlorella ellipsoidea [140]. Photosystem II (PS II) is a protein-pigment complex that is structured to catalyze the transfer of electrons from water to plastoquinone thus releasing oxygen. At the core of PS II is a dimer of two related proteins, called D1 and D2, which bind the pigments and cofactors involved in electron transfer. The D1 protein is susceptible to PAR-induced photoinhibition and PAR driven turnover of the D1 protein is rapid through synthesis via the PS II repair cycle. Both the D1 and the D2 proteins are susceptible to degradation by UV-B especially at 300 nm and there is UVR-induced turnover of these proteins to prevent the accumulation of UVR-damaged PS II. Inhibition of protein synthesis by the antibiotic streptomycin in the diatom Thalassiosira psuedonana results in a greater susceptibility to photoinhibition of carbon uptake by UV-B [141]. The repair cycle can also be damaged thus limiting the turnover of functional D1 resulting in photoinhibition of photosynthesis due to a decrease in the capacity of PS II to transfer electrons [114,142]. In the aquatic cyanobacterium, Synechococcus sp., the genes psbAII and psbAIII are induced within 15 min of moderate exposure to UV-B. This induction results in an exchange of two distinct D1 proteins, Dl:l, encoded by the constitutively-expressed psbAI, and Dl:2, encoded by psbAII and psbAIII. Under the same UVR conditions, a mutant only able to express psbAI experienced a 40% drop in photosynthesis, whereas a mutant able to constitutively express psbAII and psbAIII was able to resist exposure to UV-B [142].

10.4.2 DNA repair and DNA damage tolerance mechanisms

Although the peak absorbance of DNA is at 260 nm, the absorption spectrum of DNA follows a normal distribution that extends well into the UV-B; therefore, exposure to any of these wavelengths can result in damage to DNA. UVR-induced damage can result in direct mutagenesis as well as lethal effects due to the structural changes in DNA molecules, which can interfere in the synthesis of DNA and in the transcription by RNA resulting in errors in translation of the genetic code. The most common structural change in DNA is the formation of crosslinks between bases on the same strand of DNA, the most common photo-product of which is the formation of dimers between adjacent pyrimidine bases called cyclobutane pyrimidine dimers (CPDs). CPDs make up 50 to 80% of

UVR-induced DNA photoproducts with most of the remaining portion being made up of pyrimidine (6-4) pyrimidone dimers or 6-4 photoproducts [143]. CPDs do not cause mutagenesis as the dimers cannot base pair with other nucleotides; however, they do cause a block in DNA replication. Thus, dimer damage to DNA incapacitates its normal function of directing cellular metabolism including the replication of DNA, the transcription of genes and the synthesis of protein. There are two DNA repair pathways: photoreactivation, nucleotide excision repair and two DNA damage tolerance mechanisms: dimer bypass and recombinational repair (Figure 1). Tolerance pathways do not repair the damage but rather reduce the effect of the photoproducts on the genetic system. Photoreactivation (DNA photorepair or photoenzymatic repair) Photoreactivation is a single enzyme repair system that utilizes UV-A and blue light energy (385 to 450 nm) to monomerize the dimers formed between the pyrimidine bases [144]. The enzyme, photolyase, recognizes and binds to the CPDs in situ and splits the dimer using light energy thus restoring the bases to their native form (Figure 1). Photolyases can have either a folate- or a flavin-type chromophore, with absorption maxima between 350 and 450 nm. Excitation energy from UV-A or blue light is transferred from the chromophore to the active site that contains a flavin adenine dinucleotide (FAD), which subsequently transfers an electron to the dimer resulting in monomerization [144]. This pathway is present in viruses, mycoplasmas, and many eukaryotic organisms including protozoa, algae, higher plants, reptiles, amphibians, fish and marsupials and it is the major repair pathway in bacteria. In the marine environment, this pathway has been shown to be present as a repair mechanism for UVR-damaged DNA in microalgae [145,146], a red alga [147], marine and freshwater crustaceans [148-151], Antarctic bacteria [152], 12 species of Antarctic diatoms [153] and Antarctic zooplankton, including fish larvae [154] and a seagrass species, Halodule wrightii [155]. Organisms that depend on the repair capacity of photoreactivation also depend on the quality and quantity of light in their environment after the DNA damage has occurred. Light-independent nucleotide excision repair (dark repair)

Another pathway available for repair of DNA damage is nucleotide excision repair or dark repair, which requires an increased production of a series of DNA replication enzymes (endonuclease, DNA polymerase, exonuclease and ligase). The enzymes recognize DNA damage possibly due to distortion of the helix, incise the DNA strand at the lesion, resynthesize the correct sequence using the information from the complementary undamaged strand of DNA and DNA polymerase, excise the lesion and close the DNA strand with ligase (Figure 1). This pathway, which does not require light energy, occurs in all types of prokaryotes and eukaryotes and is a major mechanism in mammalian cells. In contrast to photoreactivation, which repairs by directly reversing DNA damage, nucleotide excision repair reverses DNA damage by replacing the lesion with

A. REPAIR 1. Photoreactivation

RECOGNITION (photolyase)

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2. Nucleotide Excision Repair RECOGNITION (endonuclease)

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INCISION (endonuclease)

B. TOLERANCE 1. Dimer bypass


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2. Recombinational Repair INCOMPLETE SYNTHESIS

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RESYNTHESIS (DNA polymerase)

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SEALING (polynucleotide ligase)

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Figure 1. Schematic presentation of (A) DNA repair mechanisms: 1. Photoreactivation also known as photoenzymatic repair, and 2. Nucleotide excision repair where the lesion damaged by exposure to UV-B is reversed (photoreactivation) or expelled (nucleotide excision repair); (B) DNA damage tolerance mechanisms: 1. Dimer bypass and 2. Recombinational repair where replication proceeds around the lesion and the gap is filled in by adenine (dimer bypass) or a homologous sequence is inserted (recombinational repair).

new nucleotides. This pathway is virtually absent in Antarctic marine diatoms [153] and found in only low levels in Antarctic marine bacteria [152] but is present in Chlamydomonas [146], Dimer bypass (translesion synthesis)

This pathway has been reported in the bacterium Escherichia coli, where gene products bind to DNA polymerase, thus altering it and resulting in insertion of adenine residues directly across from the damaged portion of the strand (Figure 1). Although, incorrect bases are inserted into the complementary strand and the damage site remains unrepaired, the advantage of dimer bypass is that it allows for DNA replication to continue. Recombinational repair (post replication repair)

In some cases, the damaged portion of DNA is bypassed during the replication phase but otherwise replication continues. The recombinational or post replication pathway then inserts a homologous complementary DNA strand into the site opposite of the dimer damage (Figure 1). The dimer damage is left unrepaired and replication continued such that the complementary strand is error-free. This type of repair is known to occur in bacteria.

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