**14. The special case of asexual bdelloid rotifers**

Bdelloid rotifers are common invertebrate animals. They are apparently obligate asexuals that reproduce by parthenogenesis. These organisms are extraordinarily resistant to ionizing radiation (Gladyshev and Meselson, 2008). This resistance appears to be a consequence of an evolutionary adaptation to survive desiccation in ephemerally aquatic habitats. Such desiccation causes extensive DNA breakage, which they are able to repair. Bdelloid primary oocytes are in the G1 phase of the cell cycle and thus lack sister chromatids. Welch et al. (2008) proposed a mechanism of repair involving interaction of non-sister co-linear chromosome pairs, which are maintained as templates for repair of DNA DSBs caused by the frequent desiccation and rehydration. Thus although these organisms apparently lack sex and meiosis, an essential feature of meiosis, HRR between non-sister homologs appears to be retained.

### **15. Conservation among eukaryotes of RecA-like proteins as key components of the HRR machinery acting during meiosis**

Sex appears to be universally based on RecA-like proteins. RecA-like proteins play a key role in HRR, and the HRR machinery and its mechanism of action appear to be highly conserved among eukaryotes. The r*ad51* and d*mc1* genes in the eukaryotic yeasts *S. cerevisiae* and *S. pombe* are orthologs of the bacterial *recA* gene. The *dmc1* gene is found in

Meiosis as an Evolutionary Adaptation for DNA Repair 373

Fig. 2. Conservation of the key components of the HRR machinery during the sexual process of transformation in bacteria and during meiosis in eukaryotes. The bacterial RecA protein or the eukaryotic RecA-like protein, Dmc1, assembles on single-stranded DNA to form a pre-synaptic filament. This filament then attaches to a duplex DNA molecule and searches for homology in its target. When the pre-synaptic molecule locates an homologous sequence in the duplex molecule, it is able to form a DNA joint. These joints are then processed

further to complete the HRR event.

many different eukaryote species, and has been reported, for instance, in the protists *Giardia*, *Trypanosoma*, *Leishmania*, *Entamoeba* and *Plasmodium* (Ramesh et al., 2005). Rad51 and Dmc1 proteins are recombinases that interact with single-stranded DNA to form filamentous intermediates called presynaptic filaments, and these filaments initiate HRR (Sauvageau et al., 2005; San Filippo et al., 2008). Dmc1 recombinase functions only during meiosis, whereas Rad51 recombinase acts in both somatic HRR and in meiosis. When it functions in meiosis, Rad51 mainly uses a sister chromosome for HRR. In contrast, Dmc1 mainly uses the non-sister homologous chromosome. The yeast Rad51 recombinase catalyzes ATP-dependent homologous DNA pairing and strand exchange, as does the bacterial RecA recombinase (Sung, 1994). The tertiary structure of the Dmc1 recombinase has an overall similarity to the bacterial RecA recombinase (Story et al., 1993). These observations suggest that the bacterial RecA that functions in the bacterial sexual process of transformation, and the yeast Rad51 and Dmc1 recombinases that act in meiosis have similar functions, consistent with the idea that meiotic recombination evolved from simpler sexual processes in bacteria

We next consider evidence that RecA orthologs play a key role in meiosis, not only in protists, but also in multicellular eukaryotes. RecA orthologs act in meiosis in a range of animals (e.g. nematodes, chickens, humans and mice) and plants (e.g. *Arabidopsis*, rice and lilies). The *rad51* gene is expressed at a high level in mouse testis and ovary, suggesting that Rad51 protein is involved in meiotic recombination (Shinohara et al., 1993). In mice, mutations in the *dmc1* gene cause sterility, failure to undergo intimate pairing of homologous chromosomes and an inability to complete meiosis (Pittman et al., 1998; Yoshida et al., 1998; see also Table 2). In the nematode *C. elegans*, resistance to DNA damage caused by X-irradiation in the meiotic pachytene nuclei depends on a RecA-like gene (Takanami et al., 2000). *RecA* gene orthologs are also expressed in chicken testis and ovary and in human testis. In humans, Dmc1, the meiosis-specific recombinase, forms nucleoprotein complexes on single-stranded DNA that promote a search for homology and carry out strand exchange, the two necessary steps of genetic recombination (Sehorn et al, 2004; Bugreev et al., 2005).

In lily plants, genes *lim15* and *rad51* are orthologs, respectively, of the *dmc1* and *rad51* genes of yeast. The lily proteins Lim15 and Rad51 colocalize on chromosomes in various stages of meiotic prophase I, and form discrete foci (Terasawa et al., 1995). The proteins of these foci are considered to participate in the search for, and pairing of, homologous sequences of DNA. In another plant, *Arabidopsis thaliana*, meiotic recombination requires Dmc1 (Couteau et al., 1999) and Rad51 (Li et al., 2004). In the rice plant, an ortholog of dmc1 is necessary for meiosis and has a key function in the pairing of homologous chromosomes (Deng and Wang, 2007).

In general, both animals and plants have RecA-like proteins that appear to have a central function in meiotic HRR. Furthermore, bacterial RecA and its animal and plant orthologs have very similar roles in the HRR events during the sexual processes of bacterial transformation and eukaryotic meiosis. In all cases, the RecA protein or RecA-like protein assembles on single-stranded DNA to form a pre-synaptic filament. This filament then attaches to a duplex DNA molecule and searches for homology in its target. When the presynaptic molecule locates an homologous sequence in the duplex molecule, it is able to form a DNA joint [Figure 2]. These joints are then processed further to complete the HRR event.

many different eukaryote species, and has been reported, for instance, in the protists *Giardia*, *Trypanosoma*, *Leishmania*, *Entamoeba* and *Plasmodium* (Ramesh et al., 2005). Rad51 and Dmc1 proteins are recombinases that interact with single-stranded DNA to form filamentous intermediates called presynaptic filaments, and these filaments initiate HRR (Sauvageau et al., 2005; San Filippo et al., 2008). Dmc1 recombinase functions only during meiosis, whereas Rad51 recombinase acts in both somatic HRR and in meiosis. When it functions in meiosis, Rad51 mainly uses a sister chromosome for HRR. In contrast, Dmc1 mainly uses the non-sister homologous chromosome. The yeast Rad51 recombinase catalyzes ATP-dependent homologous DNA pairing and strand exchange, as does the bacterial RecA recombinase (Sung, 1994). The tertiary structure of the Dmc1 recombinase has an overall similarity to the bacterial RecA recombinase (Story et al., 1993). These observations suggest that the bacterial RecA that functions in the bacterial sexual process of transformation, and the yeast Rad51 and Dmc1 recombinases that act in meiosis have similar functions, consistent with the idea that meiotic recombination evolved from

We next consider evidence that RecA orthologs play a key role in meiosis, not only in protists, but also in multicellular eukaryotes. RecA orthologs act in meiosis in a range of animals (e.g. nematodes, chickens, humans and mice) and plants (e.g. *Arabidopsis*, rice and lilies). The *rad51* gene is expressed at a high level in mouse testis and ovary, suggesting that Rad51 protein is involved in meiotic recombination (Shinohara et al., 1993). In mice, mutations in the *dmc1* gene cause sterility, failure to undergo intimate pairing of homologous chromosomes and an inability to complete meiosis (Pittman et al., 1998; Yoshida et al., 1998; see also Table 2). In the nematode *C. elegans*, resistance to DNA damage caused by X-irradiation in the meiotic pachytene nuclei depends on a RecA-like gene (Takanami et al., 2000). *RecA* gene orthologs are also expressed in chicken testis and ovary and in human testis. In humans, Dmc1, the meiosis-specific recombinase, forms nucleoprotein complexes on single-stranded DNA that promote a search for homology and carry out strand exchange, the two necessary steps of genetic recombination (Sehorn et al,

In lily plants, genes *lim15* and *rad51* are orthologs, respectively, of the *dmc1* and *rad51* genes of yeast. The lily proteins Lim15 and Rad51 colocalize on chromosomes in various stages of meiotic prophase I, and form discrete foci (Terasawa et al., 1995). The proteins of these foci are considered to participate in the search for, and pairing of, homologous sequences of DNA. In another plant, *Arabidopsis thaliana*, meiotic recombination requires Dmc1 (Couteau et al., 1999) and Rad51 (Li et al., 2004). In the rice plant, an ortholog of dmc1 is necessary for meiosis and has a key function in the pairing of homologous chromosomes (Deng and

In general, both animals and plants have RecA-like proteins that appear to have a central function in meiotic HRR. Furthermore, bacterial RecA and its animal and plant orthologs have very similar roles in the HRR events during the sexual processes of bacterial transformation and eukaryotic meiosis. In all cases, the RecA protein or RecA-like protein assembles on single-stranded DNA to form a pre-synaptic filament. This filament then attaches to a duplex DNA molecule and searches for homology in its target. When the presynaptic molecule locates an homologous sequence in the duplex molecule, it is able to form a DNA joint [Figure 2]. These joints are then processed further to complete

simpler sexual processes in bacteria

2004; Bugreev et al., 2005).

Wang, 2007).

the HRR event.

Fig. 2. Conservation of the key components of the HRR machinery during the sexual process of transformation in bacteria and during meiosis in eukaryotes. The bacterial RecA protein or the eukaryotic RecA-like protein, Dmc1, assembles on single-stranded DNA to form a pre-synaptic filament. This filament then attaches to a duplex DNA molecule and searches for homology in its target. When the pre-synaptic molecule locates an homologous sequence in the duplex molecule, it is able to form a DNA joint. These joints are then processed further to complete the HRR event.

Meiosis as an Evolutionary Adaptation for DNA Repair 375

Animals and plants have RecA-like proteins that have key functions in meiotic recombination involving homology recognition and strand exchange. The function of these eukaryotic proteins is similar to the bacterial RecA protein that acts during the bacterial sexual process of transformation, further suggesting that eukaryotic meiosis may have

DNA damages appear to be a ubiquitous and serious problem for all of life. We consider that the heightened ability of meiosis to repair such damages in the DNA to be passed on to

We thank Deborah Shelton, Denis Roze and Mike Berman for their thoughtful and very helpful comments on drafts of the manuscript. This work was supported in part by Arizona Biomedical Research Commission Grant #0803 and the Department of Veterans Affairs (VA), Veterans Health Administration, Office of Research and Development, VA Merit Review Grant 0142 of the Southern Arizona Veterans Affairs Health Care System. This work was also supported by National Science Foundation grant DEB-0742383 and the College of

Agrawal, A.P. (2006). Evolution of sex: why do organisms shuffle their genotypes? *Current* 

Aladjem, M.I., Spike, B.T., Rodewald, L.W., Hope, T.J., Klemm, M., Jaenishc, R. & Wahl,

Allers, T. & Lichten, M. (2001). Differential timing and control of noncrossover and

Ames, B.N., Shigenaga, M.K. & Hagen, T.M. (1993). Oxidants, antioxidants, and the

Andersen, S.L. & Sekelsky. J. (2010). Meiotic versus mitotic recombination: two different routes for double-strand break repair. *Bioessays,* Vol. 32, pp. 1058-1066. Avise, J.C. (2008). Clonality: *The genetics, ecology, and evolution of sexual abstinence in vertebrate* 

Baker, B.S., Boyd, J.B., Carpenter, A.T.C., Green, M.M., Nguyen, T.D., Ripoll, P. & Smith,

Barlow, C., Liyanage, M., Moens, P.B., Tarsounas, M., Nagashima, K., Brown, K.,

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G.M. (1998). ES cells do not activate p53-dependent stress responses and undergo p53-independent apoptosis in response to DNA damage. *Current Biology,* Vol. 8, pp.

degenerative diseases of aging. *Proceedings of the National Academy of Sciences USA,*

P.D. (1976). Genetic controls of meiotic recombination and somatic DNA metabolism in *Drosophila melanogaster*. *Proceedings of the National Academy of Sciences* 

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the next generation is a capability sufficient to explain its widespread occurrence.

evolved from simpler sexual processes in bacteria.

**17. Conclusion** 

**19. References** 

145-155.

**18. Acknowledgment** 

Science at the University of Arizona.

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#### **16. Summary**

Currently there is no general agreement among biologists on the adaptive function of sex. Meiosis, a key stage of the sexual cycle, involves close pairing and physical recombination and information exchange between homologous chromosomes ordinarily derived from two different parents. Fundamental to solving the problem of why sex exists is achieving an understanding of the function of meiosis.

A primitive form of meiosis was likely present early in the evolution of eukaryotes, perhaps in the single-celled ancestor of all eukaryotes that arose from ancestral bacteria over 1.5 billion years ago. Meiosis may be derived from bacterial transformation, a prokaryotic sexual process that promotes homologous recombinational repair of DNA as shown in Figure 2. Among extant single-cell eukaryotes, meiosis and facultative sex are ubiquitous. Entry into the sexual cycle ordinarily occurs in response to stressful conditions, such as oxidative stress, that tend to be associated with DNA damage. Thus meiosis may be an adaptation for dealing with such stresses and the resulting DNA damages. Consistent with this idea, exposure of eukaryotes to various DNA damaging agents increases meiotic recombination. Both in mitosis and meiosis, DNA damages caused by different exogenous agents are repaired by HRR, suggesting that DNA damages from natural sources (e.g. ROS) are also repaired by HRR. The consistent function of recombination in DNA repair across meiosis and mitosis in eukaryotes, and transformation in prokaryotes, is what we seek to understand through the repair hypothesis.

Defective HRR during meiosis causes infertility in humans and rodents, suggesting that removal of DNA damages is an essential function of meiosis. The majority of HRR events during both mitosis and meiosis are of the NCO type. NCO recombination is able to repair DNA damages from diverse sources. Furthermore NCO recombination likely occurs by synthesis-dependent strand annealing, a mechanism that involves a small exchange of information between two chromosomes but not physical exchange of DNA. Explanations of the adaptive function of meiosis that focus exclusively on crossing over, the minority of recombination events, are inadequate to explain the majority, the NCO type.

The Spo11 protein, a nuclease, produces DSBs that can initiate recombination and promote proper chromosome segregation. We speculate that Spo11 is part of a process that converts a variety of types of DNA damages to a "common currency," the DSB, which is then subject to HRR. During meiosis, DNA damages arising from a variety of sources can be repaired by HRR of the CO type, and this repair may occur independently of Spo11.

Genetic variation produced by meiotic recombination may provide a long-term benefit at the population level by reducing linkage disequilibrium and providing gene combinations on which selection can more effectively act, but the short-term adaptive benefit that maintains the machinery of meiosis is likely DNA repair. In contrast to mitosis, meiosis may allow greater accuracy in the repair of DNA damages, since double-strand damages occurring prior to DNA replication can, in principle, be accurately removed by HRR between non-sister homologous chromosomes, a process that is largely unavailable during mitosis.

Among different species, meiosis is frequently associated with outcrossing. This probably reflects the benefit of masking deleterious recessive alleles. However, numerous species that undergo meiosis are largely inbreeding or self-fertilizing. This implies that meiosis provides a benefit (accurate DNA repair) independently of the benefit of outcrossing and masking deleterious recessive alleles.

Animals and plants have RecA-like proteins that have key functions in meiotic recombination involving homology recognition and strand exchange. The function of these eukaryotic proteins is similar to the bacterial RecA protein that acts during the bacterial sexual process of transformation, further suggesting that eukaryotic meiosis may have evolved from simpler sexual processes in bacteria.

#### **17. Conclusion**

374 DNA Repair

Currently there is no general agreement among biologists on the adaptive function of sex. Meiosis, a key stage of the sexual cycle, involves close pairing and physical recombination and information exchange between homologous chromosomes ordinarily derived from two different parents. Fundamental to solving the problem of why sex exists is achieving an

A primitive form of meiosis was likely present early in the evolution of eukaryotes, perhaps in the single-celled ancestor of all eukaryotes that arose from ancestral bacteria over 1.5 billion years ago. Meiosis may be derived from bacterial transformation, a prokaryotic sexual process that promotes homologous recombinational repair of DNA as shown in Figure 2. Among extant single-cell eukaryotes, meiosis and facultative sex are ubiquitous. Entry into the sexual cycle ordinarily occurs in response to stressful conditions, such as oxidative stress, that tend to be associated with DNA damage. Thus meiosis may be an adaptation for dealing with such stresses and the resulting DNA damages. Consistent with this idea, exposure of eukaryotes to various DNA damaging agents increases meiotic recombination. Both in mitosis and meiosis, DNA damages caused by different exogenous agents are repaired by HRR, suggesting that DNA damages from natural sources (e.g. ROS) are also repaired by HRR. The consistent function of recombination in DNA repair across meiosis and mitosis in eukaryotes, and transformation in prokaryotes, is what we seek to

Defective HRR during meiosis causes infertility in humans and rodents, suggesting that removal of DNA damages is an essential function of meiosis. The majority of HRR events during both mitosis and meiosis are of the NCO type. NCO recombination is able to repair DNA damages from diverse sources. Furthermore NCO recombination likely occurs by synthesis-dependent strand annealing, a mechanism that involves a small exchange of information between two chromosomes but not physical exchange of DNA. Explanations of the adaptive function of meiosis that focus exclusively on crossing over, the minority of

The Spo11 protein, a nuclease, produces DSBs that can initiate recombination and promote proper chromosome segregation. We speculate that Spo11 is part of a process that converts a variety of types of DNA damages to a "common currency," the DSB, which is then subject to HRR. During meiosis, DNA damages arising from a variety of sources can be repaired by

Genetic variation produced by meiotic recombination may provide a long-term benefit at the population level by reducing linkage disequilibrium and providing gene combinations on which selection can more effectively act, but the short-term adaptive benefit that maintains the machinery of meiosis is likely DNA repair. In contrast to mitosis, meiosis may allow greater accuracy in the repair of DNA damages, since double-strand damages occurring prior to DNA replication can, in principle, be accurately removed by HRR between non-sister homologous chromosomes, a process that is largely unavailable

Among different species, meiosis is frequently associated with outcrossing. This probably reflects the benefit of masking deleterious recessive alleles. However, numerous species that undergo meiosis are largely inbreeding or self-fertilizing. This implies that meiosis provides a benefit (accurate DNA repair) independently of the benefit of outcrossing and masking

recombination events, are inadequate to explain the majority, the NCO type.

HRR of the CO type, and this repair may occur independently of Spo11.

**16. Summary** 

during mitosis.

deleterious recessive alleles.

understanding of the function of meiosis.

understand through the repair hypothesis.

DNA damages appear to be a ubiquitous and serious problem for all of life. We consider that the heightened ability of meiosis to repair such damages in the DNA to be passed on to the next generation is a capability sufficient to explain its widespread occurrence.

#### **18. Acknowledgment**

We thank Deborah Shelton, Denis Roze and Mike Berman for their thoughtful and very helpful comments on drafts of the manuscript. This work was supported in part by Arizona Biomedical Research Commission Grant #0803 and the Department of Veterans Affairs (VA), Veterans Health Administration, Office of Research and Development, VA Merit Review Grant 0142 of the Southern Arizona Veterans Affairs Health Care System. This work was also supported by National Science Foundation grant DEB-0742383 and the College of Science at the University of Arizona.

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**20** 

*Finland* 

**From Seed to Tree: The Functioning** 

*2Finnish Forest Research Institute, Parkano Research Unit, Parkano* 

In order to alleviate harmful effects of DNA damage and maintain genome integrity, all living organisms have developed a complex network of DNA repair mechanisms. However, the biochemical and genetic studies of DNA repair pathways have hitherto focused mostly on bacterial, yeast and mammalian systems (Sancar et al., 2004; Pan et al., 2006; Goosen & Moolenaar, 2008; Jackson & Bartek, 2009), whereas plants have been somewhat neglected in this respect. In plant cells, DNA damages can be generated "spontaneously" by reactive metabolites and by mistakes that occur during DNA replication and recombination processes or they can arise from exposure to environmental DNA damaging agents (Tuteja et al., 2001 & 2009). Plants are sessile organisms, which are continuously exposed to a wide variety of biotic and abiotic stresses, which can cause DNA damages directly or indirectly via the generation of reactive oxygen species (ROS) (Roldán-Arjona & Ariza, 2009). In plants, mutations, which initially arise in somatic cells, may also be present in gametes because plants lack a reserved germline and produce meiotic cells late in development (Walbot and Evans, 2003). However, the mutation rate in long-lived coniferous forest trees, such as pines, is not unexpectedly high, which indicates that the activities responsible for maintaining genome integrity must be efficient in somatic cells (Willyard et al., 2007). This chapter gives an overview of the special requirement of DNA repair in plants particularly from the point of view of longevity and the lifestyle of plants. We introduce the sequences of the Scots pine (*Pinus sylvestris* L.) putative *RAD51* and *KU80* genes which are involved in the repair of double-strand breaks (DSBs) by homologous recombination (HR) and non-homologous end-joining (NHEJ), respectively. The novel sequence data is used in the reconstruction of the evolutionary history of the *RAD51* and *KU80* genes in eukaryotes. In addition, the use of the HR and NHEJ pathways is demonstrated during the Scots pine seed development. From its early stages of development in the mother plant onwards, a pine seed is exposed to developmentally programmed as well as environmental stresses which are potentially damaging to the genome. Furthermore, the pine seed represents an interesting inheritance of seed tissues as well as anatomically well-described sequences of embryogenesis. Thus, we consider the pine seed to be a model system for studying the DNA repairing mechanisms, yet not solely within plants,

**1. Introduction** 

but in wider use – for eukaryotes in general.

Jaana Vuosku1,2, Marko Suokas1, Johanna Kestilä1,

Tytti Sarjala2 and Hely Häggman1

*1Department of Biology, University of Oulu, Oulu* 

**and Evolution of DNA Repair in Plants** 

particles in meiotic prophase I nuclei of *Caenorhabditis elegans*. *Journal of Radiation Research*, Vol. 44, pp. 271-276.

