**4. During mitosis and meiosis, DNA damages caused by diverse exogenous agents can be repaired by homologous recombination**

Molecular recombination (that is homologous physical exchange or informational exchange) during mitosis and meiosis functions as a DNA repair process designated homologous recombinational repair (HRR). Many of the gene products employed in mitotic HRR are also employed in recombination during meiosis. It is this consistent function of recombination across meiosis and mitosis in eukaryotes and transformation in prokaryotes that we seek to understand through the repair hypothesis. Mutants defective in HRR genes in *D. melanogaster* and yeast have reduced ability to repair DNA damages arising from a variety of exogenous sources. These mutants are also defective in recombination during meiosis. In general, loss of HRR capability causes increased sensitivity to killing by agents that harm cells primarily through induction of DNA damage. These agents are listed in Table 1. There have been no reports, that we know of, that HRR defective cells are sensitive to agents that harm cells by mechanisms other than primarily causing DNA damage.

In *D. melanogaster*, mutants defective in genes *mei-41, mei-9, hdm, spnA* and *brca2* have reduced spontaneous allelic recombination (crossing over) during meiosis and increased sensitivity to killing by exposure to numerous DNA damaging agents (Table 1). The Mei-41 protein is a structural and functional homolog of the human Atm (ataxia telangiectasia) protein (Hari et al., 1995), which plays a central role in HRR. The Mei-9 and Hdm proteins are components of a multiprotein complex that resolves meiotic recombination intermediates (Joyce et al., 2009). The SpnA protein is a homolog of yeast Rad51 (Staeva-Vieira et al., 2003), and Rad51 plays a central role in strand-exchange during HRR. The *D. melanogaster* Brca2 protein, a homolog of the human Brca2 protein that protects against breast cancer, regulates the activity of Rad51 protein in HRR. The Brca2 protein is required for HRR of DSBs during meiosis (Klovstad et al., 2008).

Meiosis as an Evolutionary Adaptation for DNA Repair 361

DNA damaging agent(s)

methanesulfonate, nitrogen mustard, benzo(s)pyrene, 2-

aminofluorene

aminofluorene

X-rays, methyl methanesulfonate

methanesulfonate, crosslinking agent 8-methoxypsoralen plus UV light

Table 1. Mutants with reduced meiotic recombination and sensitive to killing by specific

**5. In humans and rodents, defects in HRR enzymes lead to infertility, as would be expected if removal of DNA damages is an essential function of** 

About 15% of all couples in the US are infertile, and an important cause of male infertility appears to be oxidative stress during gametogenesis (Makker et al., 2009). During spermatogenesis in the mouse, DNA repair capability declines after meiosis is complete, allowing accumulation of DNA damage (Marchetti & Wyrobek, 2008). Lewis & Aitken (2005) reviewed evidence that DNA damages in the germ line of men are associated with poor semen quality, low fertilization rates, impaired pre-implantation development, increased abortion, and elevated incidence of disease in the offspring including childhood cancer. They noted that the natural causes of this DNA damage are uncertain, but the major candidate is oxidative stress. On the hypothesis that meiosis is an adaptation for DNA repair, it is expected that loss of ability to repair DNA damages during meiosis would have adverse effects, including infertility. Although the finding of such adverse effects is expected on the hypothesis that meiosis is an adaptation for repairing naturally caused DNA

methanesulfonate

*spnA* Reduced X-rays Increased Staeva-Vieira et

methanesulfonate, nitrogen mustard, benzo(s)pyrene, 2Sensitivity to killing by agent(s) Reference

Increased Baker et al., 1976; Boyd, 1978; Rasmuson, 1984

Increased Baker et al., 1976; Boyd, 1978; Rasmuson, 1984

Increased Joyce et al., 2009

Increased Klovstad et al., 2008

Increased Haynes & Kunz,

1981; Henriques & Moustacchi, 1980; Game et al., 1980

al., 2003

Organism Mutant

DNA damaging agents.

**meiosis** 

*D. melanogaster* 

gene

Meiotic recombination

*hdm* Reduced methyl

DSBs is reduced

*brca2* HRR of

*S. cerevisiae rad52* Reduced X-rays, methyl

*mei-41* Reduced X-rays, UV, methyl

*mei-9* Reduced X-rays, UV, methyl

acetyl-

acetyl-

In *S. cerevisiae,* numerous mutant genes have been identified that confer sensitivity to radiation and/or genotoxic chemicals (Haynes & Kunz, 1981). Several of these mutant genes are also defective in meiotic recombination. For instance, the *rad52* gene is required for meiotic recombination (Game et al., 1980) as well as for mitotic recombination (Malone & Esposito, 1980). Mutants defective in the *rad52* gene are sensitive to killing by several DNA damaging agents (Table 1). Diploid cells of *S. cerevisiae* are able to repair DNA DSBs introduced by ionizing radiation, and this ability is lost in mutant strains defective in the *rad52* gene (Resnick & Martin, 1976). The Rad52 protein promotes the DNA strand exchange reaction of recombination during meiosis and mitosis (Mortensen et al., 2009).

Taken as a whole, these findings indicate that the products of genes *mei-41, mei-9*, *hdm*, *spnA, and brca2* in *D. melanogaster* and the *rad52* gene of yeast are required in meiosis for recombination and in somatic cells for HRR of potentially lethal DNA damages. Since the gene products that function in mitotic HRR are able to repair DNA damages from different sources, it can be reasonably assumed that these genes serve a similar DNA repair function during recombination in meiosis.

In the nematode *C. elegans* gonad, oocyte nuclei in the pachytene stage of meiosis, the stage in which HRR occurs, are hyper-resistant to X-ray irradiation compared to oocytes in the subsequent diakinesis stage of meiosis (Takanami et al., 2000). This hyper-resistance depends on expression of gene c*e-rdh-51*, a homolog of yeast *rad51* and *dmc1* that play a central role in meiotic HRR. Meiotic pachytene nuclei are also more resistant to heavy ion particle irradiation than the subsequent meiotic diplotene or diakinesis stages (Takanami et al., 2003). This resistance also depends on the *ce-rdh-51* gene, as well as on gene *ce-atl-1*. *ce-atl-1* is related to *atm* (ataxia –telangiectasia mutated), a gene necessary for repair of DSBs by HRR.

Coogan & Rosenblum (1988) measured repair of DSBs following γ-irradiation of rat spermatogenic cells during successive stages of germ cell formation. The stages were spermatagonia and preleptotene spermatocytes, pachytene spermatocytes and spermatid spermatocytes. The greatest repair capability was observed in pachytene, the stage of meiosis when HRR occurs. These findings indicate that HRR of γ-ray-induced DSBs occurs during meiosis. Several mammalian germ cell stages, including pachytene spermatocytes, produce levels of reactive oxygen species (ROS) sufficient to cause oxidative stress (Fisher & Aitkin, 1997). This observation suggests that HRR during meiosis may also remove DNA damages caused by natural endogenously produced ROS.

The results reviewed in this section indicate that, in both meiosis and mitosis, DNA damages caused by different exogenous agents are repaired by HRR, suggesting that DNA damages from natural endogenous sources (e.g. ROS) are similarly repaired. In general, DNA damage appears to be a fundamental problem for life. As noted by Haynes (1988), DNA is composed of rather ordinary molecular subunits, which are not endowed with any peculiar kind of quantum mechanical stability. He observed that its very "chemical vulgarity" makes DNA subject to all the "chemical horrors" that might befall any such molecule in a warm aqueous medium. The average amount of oxidative DNA damage occurring per cell per day is estimated to be about 10,000 in humans, and in rat, with a higher metabolic rate, about 100,000 (Ames et al., 1993). Most of these damages affect only one strand of the DNA, but a fraction, about 1-2%, are double-strand damages such as DSBs (Massie et al., 1972). These damages can be repaired accurately by HRR.

In *S. cerevisiae,* numerous mutant genes have been identified that confer sensitivity to radiation and/or genotoxic chemicals (Haynes & Kunz, 1981). Several of these mutant genes are also defective in meiotic recombination. For instance, the *rad52* gene is required for meiotic recombination (Game et al., 1980) as well as for mitotic recombination (Malone & Esposito, 1980). Mutants defective in the *rad52* gene are sensitive to killing by several DNA damaging agents (Table 1). Diploid cells of *S. cerevisiae* are able to repair DNA DSBs introduced by ionizing radiation, and this ability is lost in mutant strains defective in the *rad52* gene (Resnick & Martin, 1976). The Rad52 protein promotes the DNA strand exchange reaction of recombination during meiosis and mitosis (Mortensen

Taken as a whole, these findings indicate that the products of genes *mei-41, mei-9*, *hdm*, *spnA, and brca2* in *D. melanogaster* and the *rad52* gene of yeast are required in meiosis for recombination and in somatic cells for HRR of potentially lethal DNA damages. Since the gene products that function in mitotic HRR are able to repair DNA damages from different sources, it can be reasonably assumed that these genes serve a similar DNA repair function

In the nematode *C. elegans* gonad, oocyte nuclei in the pachytene stage of meiosis, the stage in which HRR occurs, are hyper-resistant to X-ray irradiation compared to oocytes in the subsequent diakinesis stage of meiosis (Takanami et al., 2000). This hyper-resistance depends on expression of gene c*e-rdh-51*, a homolog of yeast *rad51* and *dmc1* that play a central role in meiotic HRR. Meiotic pachytene nuclei are also more resistant to heavy ion particle irradiation than the subsequent meiotic diplotene or diakinesis stages (Takanami et al., 2003). This resistance also depends on the *ce-rdh-51* gene, as well as on gene *ce-atl-1*. *ce-atl-1* is related to *atm* (ataxia –telangiectasia mutated), a gene necessary for repair of

Coogan & Rosenblum (1988) measured repair of DSBs following γ-irradiation of rat spermatogenic cells during successive stages of germ cell formation. The stages were spermatagonia and preleptotene spermatocytes, pachytene spermatocytes and spermatid spermatocytes. The greatest repair capability was observed in pachytene, the stage of meiosis when HRR occurs. These findings indicate that HRR of γ-ray-induced DSBs occurs during meiosis. Several mammalian germ cell stages, including pachytene spermatocytes, produce levels of reactive oxygen species (ROS) sufficient to cause oxidative stress (Fisher & Aitkin, 1997). This observation suggests that HRR during meiosis may also remove DNA

The results reviewed in this section indicate that, in both meiosis and mitosis, DNA damages caused by different exogenous agents are repaired by HRR, suggesting that DNA damages from natural endogenous sources (e.g. ROS) are similarly repaired. In general, DNA damage appears to be a fundamental problem for life. As noted by Haynes (1988), DNA is composed of rather ordinary molecular subunits, which are not endowed with any peculiar kind of quantum mechanical stability. He observed that its very "chemical vulgarity" makes DNA subject to all the "chemical horrors" that might befall any such molecule in a warm aqueous medium. The average amount of oxidative DNA damage occurring per cell per day is estimated to be about 10,000 in humans, and in rat, with a higher metabolic rate, about 100,000 (Ames et al., 1993). Most of these damages affect only one strand of the DNA, but a fraction, about 1-2%, are double-strand damages such as DSBs

damages caused by natural endogenously produced ROS.

(Massie et al., 1972). These damages can be repaired accurately by HRR.

et al., 2009).

DSBs by HRR.

during recombination in meiosis.


Table 1. Mutants with reduced meiotic recombination and sensitive to killing by specific DNA damaging agents.
