BAMBÚ

S. cerevisiae and human RadSI proteins

1 33 E. coli RecA 240 352 1 74 154 S. cerevisiae Rad51 374 400 96 H. sapiens Rad51 314 339

B

E. coli

RecA

ScRad51

» HsRad51

Figure 1.11. Sequence comparison of RecA, S. cerevisiae and human RadSl. (A) Schematic illustration of the domain structure of these proteins. The conserved core domain is shown in red, N-terminal sequences conserved between S. cerevisiae and hum an RadSl are striped.

(B) Three way comparison of RecA with its homologues. The percentages for identical plus similar amino acids within the core domains are indicated. Figure adapted from Baumann and West, 1998.

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highly flexible structure and might be involved in protein-protein interactions (Brendel et ah, 1997; Ogawa et ah, 1993).

Screening hum an databases w ith the hRadSl sequence led to the identification of two additional genes with significant hom ology to hRAD51, that w ere hence nam ed hRADSlB and hRADSlC (Albala et a l, 1997; Dosanjh et al., 1998). H um an RAD51B was also identified by two other groups and is referred to as R51H2 and hRECl (Cartwright et a l, 1998a; Rice et al, 1997). Hom ology to hRadSl was also found in two hum an genes {XRCC2 and XRCC3) that complem ent the mitomycin C sensitivity of the rodent cell lines irsl and irslSF respectively (Cartwright et al, 1998b; Liu et a l, 1998). The m utant cell lines are characterised by their sensitivity to a variety of DNA dam aging agents and a high frequency of spontaneous and m utagen-induced chrom osomal aberrations (Fuller and Painter, 1988; Tebbs et al, 1995). Since irsl and irslSF are highly sensitive to DNA cross-linking agents a specific role for Xrcc2 and XrccS in the repair of these lesions has been suggested (Caldecott and Jeggo, 1991).

The existence of a whole family of Rad51-like proteins in m am m alian cells raises the possibility that they function in related but distinct pathw ays of hom ologous recombination. Alternatively, these proteins m ight act together as a functional unit. This latter possibility is supported by genetic and biochemical analysis in S. cerevisiae, where two proteins, ScRad55 and ScRad57, show sequence hom ology w ith ScRadSl (Kans and Mortimer, 1991; Lovett, 1994). M utations in the RAD55 and RAD57 genes give rise to a cold sensitive phenotype that is suppressed by the overexpression of RadSl a n d /o r Rad52, indicating that RadSS and Rad57 may act by stabilising a complex containing RadSl and RadS2. Consistent with this hypothesis, direct protein interactions have been dem onstrated between RadSl and RadSS, as well as betw een RadSS and RadS7 (Hays et al, 1995; Johnson and Symington, 199S). The m ost compelling evidence for functional interactions betw een RadSl, RadSS and RadS7 comes from in vitro recombination assays w here the concerted action of

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the three proteins can lead to strand exchange under conditions w here ScRP-A inhibits RadSl-ssDNA filament formation in the absence of Rad55 and Rad57 (Sung, 1997b).

It is currently unclear whether the hum an RadSlB, RadSlC, Xrcc2 and XrccS proteins are functional homologues of yeast Rad55/57. Sequence com parison show ed that none of the hum an hRad51-like proteins is m ore closely related to ScRad55 or ScRad57 than to ScRadSl. However, two hybrid interactions have been observed between hRadSl and XrccS, RadSlB and hRadSlC and betw een XrccS and hRadSlC indicating that corresponding functional interactions may exist (Dosanjh et ah, 1998; Liu et ah, 1998).

D m cl - a m eiosis specific RecA/RadSl homologue

At the same time as the homology between ScRadSl and E. coli RecA w as noted, a second RecA homologue, Dmcl, was identified in S. cerevisiae. DM Cl (disrupted meiotic cDNA) is a meiosis specific gene that is induced during prophase of meiosis I (Bishop et al, 1992). Mutations in ScDMCl cause defects in the processing of meiotic DSBs and delays in synaptonem al complex form ation (Bishop et a l, 1992; Rockmill et al, 1995b; Xu et a l, 1997). The sequence homology between ScDmcl and E. coli RecA is 50% over the core domain. ScDmcl and ScRadSl colocalise on synapsed regions of hom ologous chromosomes (Bishop, 1994). Interestingly, the localisation of ScDmcl on meiotic chromosomes is dependent on ScRadSl and is not observed in a radSl deletion strain. In contrast, ScRadSl foci form in the absence of ScDmcl b u t persist for longer than observed in wild-type cells, indicating that the two proteins m ight be involved during sequential stages of recombination reactions.

Homologues of ScDmcl have been isolated from lily, mouse and hum an cDNA libraries (Habu et al, 1996; Kobayashi et al, 1993; Sato et a l, 199Sa; Sato et al, 199Sb). The lily Dm cl homologue (LimlS) has been show n to colocalise w ith the lily RadSl protein in meiotic cells (Terasawa et al, 1995). Antibodies capable

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of reacting w ith RadSl and LimlS were found to label early recombination nodules supporting a role for the two proteins in meiotic recombination (A nderson et ah, 1997).

Consistent w ith a meiosis specific function of DM Cl transcription of the m urine DMCl gene is limited to testis and ovary (Habu et a l, 1996). D isruption of the mouse DMCl gene led to sterility in hom ozygous m utant males and females (Pittman et ah, 1998; Yoshida et ah, 1998). Similar to the phenotype observed in S. cerevisiae, gametogenesis in these mice arrested during the first meiotic division. Recombinant hum an Dmcl was recently purified from E. coli (Li et a l, 1997). The protein has DNA-dependent ATPase activity and prom otes strand exchange reactions between oligonucleotide substrates.

Structural and functional analysis of S. cerevisiae and hu m an R adSl

Initial biochemical studies and electron microscopic analysis confirmed that S. cerevisiae and hum an RadSl are indeed homologues of the E. coli RecA protein. ScRadSl was found to form nucleoprotein filaments, in which the DNA is underw ound and extended to a similar degree as observed in RecA nucleoprotein filaments (Ogawa et ah, 1993). Indeed, the low resolution structures of RecA and ScRadSl filaments, based on Fourier transform ation of filament images, were strikingly similar. Remarkable similarities w ere also observed in a comparison of nucleoprotein filaments form ed by hum an RadSl and E. coli RecA protein (Benson et ah, 1994).

However, despite the obvious conservation betw een the eukaryotic RadSl proteins and RecA, biochemical studies also indicated significant differences. RecA preferentially binds to single-stranded or partially single­ stranded DNA and strand exchange occurs between an extended nucleoprotein filament and naked duplex DNA (see above). In contrast, the yeast and hum an RadSl proteins exhibit similar affinities for single- and double-stranded DNA [Shinohara, 1992 #2729; Benson, 1994]. These differences are significant, since

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studies on RecA suggest that DNA strand exchange cannot occur betw een two nucleoprotein filaments (Roca and Cox, 1990).

Like RecA, ScRadSl was found to hydrolyse ATP in the presence of DNA (Sung, 1994). However, compared to its bacterial homologue, the rate of ATP hydrolysis observed in the presence of ScRadSl was m ore than 40-fold lower. W eak DN A-dependent ATPase activities were also reported for hRadSl and X/RadSl.l'* (Benson et ah, 1994; Maeshima et ah, 1996).

Hom ologous pairing and strand exchange activity was dem onstrated for ScRadSl w hen the protein was preincubated w ith ssDNA, followed by the addition of homologous linear duplex DNA (Sung, 1994). By staging the reactions in this way, nucleoprotein filaments formed only on ssDNA and hom ologous pairing could occur between RadSl nucleoprotein filaments and naked duplex DNA. In contrast, pairing activity was not observed w hen RadSl filaments were first formed on duplex DNA, nor w hen reactions were carried out betw een two nucleoprotein filaments (Sung and Robberson, 199S). After the hom ologous pairing activity of hRadSl, described in Chapter 3 to S, had been published, similar activities were also reported by others for Xenopus laevis and hu m an RadSl using oligonucleotide substrates (Gupta et ah, 1997; M aeshima et ah, 1996).

Strand exchange reactions catalysed by ScRadSl require the presence of the yeast single-stranded DNA-binding protein ScRP-A (Sung, 1994). The effects of ScRP-A on the ATPase and strand exchange activities of ScRadSl w ere studied concomitantly w ith my analysis of the role of hum an RP-A in hRadSl- m ediated strand transfer reactions (Sugiyama et ah, 1997 and this work). Sugiyam a's w ork led to the conclusion that the m ain function of ScRP-A in ScRadSl-mediated strand exchange reactions is the rem oval of DNA secondary structures.

* XIRAD51.1 is one of tw o RAD5Î genes identified in Xenopus laevis. The amino acid sequences of X /R adSl.l and X/Rad51.2 are 98.2% identical (Maeshima et al., 1995).

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In reactions between single-stranded circular and partially hom ologous linear duplex DNA, ScRadSl prom oted joint molecule form ation w ith 3' to 5' polarity, opposite to the polarity observed w ith RecA (Sung and Robberson, 1995). However, this work has been challenged by a report suggesting that ScRadSl prom otes strand exchange w ith 3' to S' and S' to 3' polarity, depending on the structure of the DNA end at which pairing is initiated (Namsaraev and Berg, 1997). Clearly, the subject requires further analysis to determ ine w hether 3' or S' term ini are preferred substrates in the initiation of hom ologous pairing and w hether progressive strand transfer occurs w ith a defined polarity.

Expression pattern and cellular localisation of RadSl proteins

Analysis of the RadSl expression pattern in chicken and mice revealed high levels of RadSl mRNA in thym us, spleen, ovary and testis, indicating a role in proliferating cells and cells undergoing meiosis (Bezzubova et ah, 1993; Morita et ah, 1993; Shinohara et ah, 1993). W hen prim ary hum an B-cells were cultured w ith lipopolysaccharide to stimulate class switch recombination, hRadSl protein levels were found to increase dramatically (Li et ah, 1996). Immunofluorescence microscopy using anti-hRadSl antibodies revealed that hR adSl localises to small and discrete sites in the nucleus ('nuclear dots') and is largely absent from the cytoplasm. These results m ight indicate an involvem ent of hRadSl in class switch recombination.

Interestingly, 'nuclear dots' were also observed during meiotic prophase in S. cerevisiae and lily, w here RadSl colocalises w ith D m cl (Bishop, 1994; Terasawa et ah, 199S). Im munoelectron microscopy show ed that these foci coincide w ith the presence of early recombination nodules, proteinaceous structures formed at sites of meiotic recombination (Anderson et ah, 1997). Similarly, RadSl foci on synapsed regions of homologous chromosomes were observed in m ouse and hum an spermatocytes (Barlow et ah, 1997; H aaf et ah, 1995', Plug et ah, 1996).

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Support for a role of m ammalian RadSl proteins in DNA repair was given by immunofluorescence studies on cultured hum an somatic cells following treatm ent with methyl methanesulfonate, a lethal dose of UV-C or y- irradiation. Simultaneous analysis of BrdUrd incorporation and the distribution of RadSl revealed that RadSl foci form in nearly all cells undergoing repair DNA synthesis (Haaf et a l, 199S). Taken together the spatial and tem poral expression pattern of RadSl in a variety of species w as found to be consistent w ith a direct role in homologous recombination and DNA repair.

An essential role for hRadSl in cell proliferation

In light of the viability of yeast radSl m utants, it w as surprising to find that radSl'' knockout mice died early in embryonic developm ent and that radSl'' cell lines could not be established (Lim and Hasty, 1996; Tsuzuki ei al, 1996). Analysis of the hom ozygous m utant embryos revealed that death occurred after 7.5 days of embryonic development, following a rapid decrease in cell proliferation and increased cell death. The possibility that this phenotype could be due to a defect in DNA repair is supported by the observation that blastocysts of radSl'' embryos are hypersensitive to y-irradiation (Lim and H asty, 1996). Recently, a chicken cell line was generated in which RAD51 w as u n d er the control of a repressible prom oter (Sonoda et al, 1998). Inhibition of RadSl expression resulted in chromosome breaks, cell cycle arrest and cell death, establishing an essential role for RadSl in cell proliferation and genome maintenance.

Consistent w ith a role for RadSl in the repair of DNA dam age at certain stages during the cell cycle, expression levels of mouse and hum an RadSl were found to fluctuate (Chen et al, 1997; Flygare et al, 1996; Yamamoto et a l, 1996). Expression was lowest in G JG^, increased in S phase and reached a m axim um in Gy The use of actinomycin D to inhibit RNA synthesis dem onstrated th at the RAD51 gene is transcriptionally regulated (Flygare et a l, 1996). These results are

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in agreem ent w ith a role for RadSl in the repair of DSBs occurring during replication.

Rad54 is required for X-ray resistance in mammalian cells

Another member of the S. cerevisiae RAD52 epistasis group, for which hom ologues have been identified in higher eukaryotes, is RAD54 (Kanaar et al, 1996). Rad54 is a member of the Snf2/Swi2 family of DNA dependent ATPases. M embers of this family have been implicated in various aspects of DNA m etabolism such as transcription, recombination and repair (Eisen et a l, 1995). H om ozygous rad54'^' mouse and chicken cell lines have been established and characterised (Bezzubova et al, 1997; Essers et a l, 1997). The frequency of hom ologous recombination is severely reduced in both m utant cell lines, and m ore im portantly, both cell lines are sensitive to ionising radiation and DSB- inducing agents. These observations are reminiscent of the phenotype seen in rad54 m utant yeast strains and therefore indicate that the roles of Rad54 in hom ologous recombination and recombinational repair have been conserved from yeast to vertebrates.

ScRad54 interacts w ith ScRadSl in vitro and in vivo (Clever et a l, 1997; Jiang et al, 1996) and has been shown to stimulates the form ation of joint molecules w hen added to a strand exchange reaction containing ScRadSl (Petukhova et a l, 1998). However, the mechanism of this stim ulation and its significance in vivo is currently unclear. A part from direct interactions w ith hRadSl little is currently known about the biochemical activities of hum an RadS4 (Golub et al, 1997). Based on the function of other m em bers of the Snf2/Swi2 family, hRadS4 has been suggested to play a role in chrom atin rem odelling during homologous recombination (Kanaar and Hoeijmakers, 1997). O ther m odels include an involvement in the turnover of the RadSl filament following heteroduplex formation or a direct role in branch m igration.

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M ultiple functions for RadSl?

The possibility that hRadSl might be involved in processes other than hom ologous recombination has been discussed following the identification of num erous proteins believed to interact with hRadSl and the observation that RadSl is an essential gene in mammals but not in yeast. Over the last few years, hR adSl was found to interact with the ubiquitin-conjugating enzyme Ubc9 (Kovalenko et ah, 1996), the ubiquitin-like protein U bll (Shen et ah, 1996a), the tum our suppressors pS3, Brcal and Brca2 (Scully et ah, 1997; Sharan et ah, 1997; Stürzbecher et ah, 1996) and the tyrosine kinase c-Abl (Yuan et ah, 1998). Furtherm ore, RadSl was isolated as part of a large complex containing RNA polym erase II and DNA polymerase e, as well as com ponents of the non- hom ologous end-joining and nucleotide excision repair machineries (M aldonado et ah, 1996).

Given the results described above, one could imagine hRadSl to be involved in homologous recombination, non-hom ologous end-joining, transcription, replication and protein turnover. Of course, some of these protein contacts m ight reflect regulatory interactions required to co-ordinate different cellular processes and others m ight not be significant in vivo. In either case, the confusing array of hRadSl interacting proteins em phasised the im portance of biochemical studies to further elucidate the enzymatic functions of hRadSl.

In this thesis the purification of hRadSl and the characterisation of its biochemical properties is described. Hum an RadSl was show n to bind to single- and double-stranded DNA and display D N A -dependent ATPase activity. Conditions were established under which hRadSl prom otes hom ologous pairing and strand exchange reactions betw een plasm id-sized DNA substrates. Further studies demonstrated that hRP-A and hRadS2 can both stimulate the activity of hRadSl and the mechanisms of these interactions w ere investigated.

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