U.S. patent application number 10/912594 was filed with the patent office on 2005-06-02 for compositions and methods using eukaryotic rad5.
Invention is credited to Golub, Efim Ilya, Radding, Charles Meyer, Reddy, Gurucharan.
Application Number | 20050118614 10/912594 |
Document ID | / |
Family ID | 26704474 |
Filed Date | 2005-06-02 |
United States Patent
Application |
20050118614 |
Kind Code |
A1 |
Reddy, Gurucharan ; et
al. |
June 2, 2005 |
Compositions and methods using eukaryotic Rad5
Abstract
The invention relates to complexes of eukaryotic Rad52 protein
and nucleic acids, and methods of using the complexes.
Inventors: |
Reddy, Gurucharan; (Redwood
City, CA) ; Golub, Efim Ilya; (New Haven, CT)
; Radding, Charles Meyer; (Hamden, CT) |
Correspondence
Address: |
DORSEY & WHITNEY LLP
INTELLECTUAL PROPERTY DEPARTMENT
4 EMBARCADERO CENTER
SUITE 3400
SAN FRANCISCO
CA
94111
US
|
Family ID: |
26704474 |
Appl. No.: |
10/912594 |
Filed: |
August 4, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10912594 |
Aug 4, 2004 |
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09434196 |
Nov 4, 1999 |
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09434196 |
Nov 4, 1999 |
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08781329 |
Jan 10, 1997 |
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5989879 |
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60029055 |
Oct 24, 1996 |
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Current U.S.
Class: |
435/6.11 ;
435/6.1; 435/91.2; 514/44R |
Current CPC
Class: |
C07K 14/47 20130101;
C12Q 1/6813 20130101; C12Q 1/6832 20130101; C12N 15/10
20130101 |
Class at
Publication: |
435/006 ;
514/044; 435/091.2 |
International
Class: |
A61K 048/00; C12Q
001/68; C12N 009/12; C12P 019/34 |
Claims
We claim:
1. A composition comprising a complex of a first single stranded
nucleic acid and isolated Rad52 protein from a higher
eukaryote.
2. A composition according to claim 1 wherein said complex is
capable of mediating the annealing of said first nucleic acid to a
complementary second single stranded nucleic acid.
3. A composition according to claim 1 wherein said Rad52 protein is
a mammalian Rad52 protein.
4. A composition according to claim 1 wherein said Rad52 protein is
a human Rad52 protein.
5. A composition according to claim 1 further comprising a second
single stranded nucleic acid complexed with isolated Rad52 protein
from a higher eukaryote.
6. A composition according to claim 5 wherein said second nucleic
acid is complementary to said first nucleic acid.
7. A composition according to claim 1 further comprising a double
stranded nucleic acid comprising second and third single stranded
nucleic acids, wherein both said first and said third nucleic acids
are complementary to said second nucleic acid.
8. A method of making double stranded nucleic acid comprising
contacting: a) a first single stranded nucleic acid; b) a second
single stranded nucleic acid, wherein said first and second nucleic
acids are complementary; and c) isolated Rad52 protein from a
higher eukaryote; under conditions whereby said Rad52 mediates
annealing of said first and second nucleic acids.
9. A method according to claim 8, wherein one or both of said
nucleic acids are complexed with said isolated Rad52 protein prior
to said contacting.
10. A method according to claim 8, wherein said annealing is done
in the absence of Mg.sup.t2 and cofactors.
11. A method of accomplishing strand exchange comprising
contacting: a) a first single stranded nucleic acid; b) a double
stranded nucleic acid comprising second and third single stranded
nucleic acids, wherein both said first and third nucleic acids are
complementary to said second nucleic acid; and c) isolated Rad52
from a higher eukaryote; under conditions whereby said Rad52
mediates the annealing of said first nucleic acid to said second
nucleic acid, such that said third nucleic acid is displaced.
12. A method according to claim 11 wherein any or all of said
nucleic acids are complexed with said Rad52 prior to said
contacting.
13. A method according to claim 11, wherein said annealing is done
in the absence of Mg.sup.t2 and cofactors.
14. A method of screening for a bioactive agent involved in
homologous recombination comprising: a) contacting: i) a candidate
bioactive agent; ii) a first single stranded nucleic acid; and iii)
isolated Rad52 protein from a higher eukaryote; and b) screening
for binding of said candidate and said Rad52 to said nucleic
acid.
15. A method according to claim 14 wherein said first nucleic acid
and said isolated Rad52 are complexed prior to the addition of said
candidate agent.
16. A method of screening for a bioactive agent involved in
homologous recombination comprising: a) adding: i) a candidate
bioactive agent; ii) a first single stranded nucleic acid; and iii)
isolated Rad52 protein from a higher eukaryote to form a mixture;
and b) screening said mixture for altered biological activity, when
compared to the biological activity of said composition in the
absence of said candidate.
17. A method according to claim 16 wherein said first nucleic acid
and said isolated Rad52 are complexed prior to the addition of said
candidate agent.
Description
FIELD OF THE INVENTION
[0001] The invention relates to complexes of eukaryotic Rad52
protein and nucleic acids, and methods of using the complexes.
BACKGROUND OF THE INVENTION
[0002] Repair of DNA damage is critical for the maintenance of
genome integrity and cell survival. Living organisms have developed
different pathways of DNA repair to deal with various types of DNA
damage. One of the pathways of repairing DNA damage is through
homologous recombination. The RecA protein of E. coli has been
shown to be important for homologous recombination, and a great
deal is known about this process in E. coli.
[0003] Although eukaryotic cells have RecA homologue(s), the
mechanism of homologous recombination in vivo are poorly understood
in eukaryotes. There is no evidence yet that eukaryotic cells
employ RecA-like recombination mechanisms in vivo.
[0004] In the yeast S. cerevisiae, three epistasis groups of DNA
damage-repair genes have been identified (Friedberg, E. C., Siede,
W. and Cooper, A. J. (1991) in The Molecular and Cellular Biology
of the yeast Saccharomyces (Broach, J. R., Pringle, J. R., Jones,
E. W., eds) pp 147-192, Cold Spring Harbor Laboratory press,
Plainview, N.Y.; Game, J. (1983) in Yeast Genetics: Fundamental and
Applied Aspects (Spencer, J. F. T., Spencer, D., and Smith., A. R.
W., eds) pp 105-137, Springer-verlag, New York.). The Rad52
epistasis group, which is mainly responsible for double-strand
break (DSB) repair contains several genes: Rad50-Rad57, MRE11 and
XRS2. Among these genes, mutations in Rad51, Rad52 and Rad54 cause
the most severe and pleiotropic defects (Game, J. (1983) in Yeast
Genetics: Fundamental and Applied Aspects (Spencer, J. F. T.,
Spencer, D., and Smith., A. R. W., eds) pp 105-137,
Springer-verlag, New York.; Ajimura, M., Leem, S. H. and Ogawa, H.
(1993) Genetics 133, 51-66; Ivanov, E. L., Korolev, V. G. and
Fabre, F. (1992) Genetics 132, 651-664; Petes, T. D., Malone, R. E.
and Symington, 1. S. (1991) 407-521.). Yeast strains lacking a
functional Rad52 gene are extremely X-ray sensitive and deficient
in mitotic and meiotic recombination (Resnick, M. A. (1969)
Genetics 62, 519-531). It was reported recently that the
overexpression of human Rad52 (HsRad52) conferred enhanced
resistance to gamma rays and induced homologous intrachromosomal
recombination in cultured monkey cells (Park, M. S. (1995) J. Biol.
Chem. 270, 15467-15470). Mutations in different regions of Rad52
often result in different phenotypes (Boundy-Mills, K. and
Livingston, D. M. (1993) Genetics 133, 39-49). It is proposed that
the product of Rad52 gene is not required for the initiation of
recombination, but is essential for an intermediate stage following
the formation of DSBs but before the appearance of stable
recombinants (Shinohara, A., Ogawa, H. and Ogawa, T. (1992) Cell
69, 457470).
[0005] In the yeast S. cerevisiae, the major pathway of
double-strand break repair is through gap repair, leading to gene
conversion that may be associated with a crossover of flanking
markers (Szostak, J. W., Orr-weaver, T. L., Rothstein, R. J. and
Stahl, F. W. (1983) Cell 33, 25-35). Both Rad51 and Rad52 are
important for the repair of breaks by this mechanism. However, gene
conversion is only one of several homologous and non-homologous
recombination pathways that are found in yeast and mammalian cells
to repair chromosomal DSBs (Haber, J. E. (1995) BioEssays 17,
609-620). Based on transformation experiments in mammalian cells
and in Xenopus oocytes, single-strand annealing, a non-conservative
mechanism, has been proposed as an alternate pathway to repair
double-strand breaks. (Fishman-Lobell, J., Rudin, N. and Haber, J.
E. (1992) Mol. Cell. Biol. 12, 1292-1303; Lin, F.-L. M., Sperle, K.
and Sternberg, N. (1990) Mol. Cell. Biol. 10, 103-112; Maryon, E.
and Carrol, D. (1991) Mol Cell. Biol. 11, 3278-3287; Jeongyu, S. J.
and Carrol, D. (1992) Mol. Cell. Biol. 12, 112-119.). Rad52 appears
to be important for all homologous recombination events including
gene conversion and single-strand annealing.
[0006] Despite the importance of Rad52 for homologous recombination
the repair of chromosomal breaks, there is very little information
available on the biochemistry of Rad52 protein. Homologs of Rad52
gene have been found in several eukaryotic organisms including
yeast, mouse, chicken and human (see Park, J. Biol. Chem.
270:15467-15470 (1995); Muris et al., Mutation Res., DNA Repair
315:295-305 (1994); Bezzubova et al., Nucleic Acid Res.
21(25):5945-5949 (1993); Bendixen et al., Genomics 23:300-303
(1994); Shen et al., Genomics 25:199-206 (1995)).
[0007] Sequence analysis has revealed that N-terminal amino acid
sequence of Rad52 protein is highly conserved while the C-terminal
region is less conserved (Bezzubova, O., Schmidt, H., Ostermann,
K., Heyer, W. D. and Buerstedde, J.-M. (1993) Nucleic Acids Res.
21, 5945-5949; Muris, D. F. R., Vreeken, K., Carr, A. M.,
Broughton, B. C., Lehman, A. R., Lohman, P. H. M. and Pastnik, A.
(1993) Nucleic Acids Res. 21, 4586-4591; Bendixen, C., Sunjevaric,
I., Bauchwitz, R. and Rothstein, R. (1994) Genomics 23, 300-303;
Shen, Z., Denison, K., Lobb, R., Gatewood, J. M. and Chen, D. J.
(1995) Genomics 25, 199-206).
[0008] It has been shown that Rad52 protein interacts through its
C-terminal domain with the N-terminal domain of Rad51 protein in a
species specific manner (Milne, G. T. and Weaver, D. T. (1993)
Genes & Dev 7, 1755-176514; Donovan, J. W., T., M. G. and
Weaver, D. T. (1994) Genes & Dev 8, 2552-2562; Shen, Z., Cloud,
K. G., Chen, D. J. and Park, M. S. (1996) J. Biol. Chem. 271,
148-152). Similarly, a specific interaction with RPA has also been
shown to be important for homologous recombination (see Park et
al., J. Biol. Chem. 271(31):18996-19000 (1996)).
[0009] Yeast Rad52 protein has been reported to bind to both single
and double-stranded DNA and carries out annealing of homologous
single-stranded DNA. (Ogawa, T., Shinohara, A., Nabetani, A.,
Ikeya, T., Yu, X., Egelman, E. H. and Ogawa, H. (1993) Cold Spring
Harbor Symp Quant. Biol. 58, 567-576.). Besides the report that
human Rad52 protein interacts with human Rad51 protein and RPA,
there are no other biochemical reports on the function of human
Rad52 protein to date.
SUMMARY OF THE INVENTION
[0010] The present invention provides compositions comprising
complexes of a first single stranded nucleic acid and isolated
Rad52 protein from a higher eukaryote. The compositions are capable
of mediating the annealing of said first nucleic acid to a
complementary second single stranded nucleic acid.
[0011] Further provided are compositions further comprising a
second single stranded nucleic acid complexed with isolated Rad52
protein from a higher eukaryote. The first and second nucleic acids
may be complementary.
[0012] Additionally provided are compositions further comprising a
double stranded nucleic acid comprising second and third single
stranded nucleic acids. Both the first and third nucleic acids are
complementary to the second nucleic acid.
[0013] Further provided are methods of making double stranded
nucleic acid. The method comprises the step of contacting a) a
first single stranded nucleic acid; b) a second single stranded
nucleic acid, and c) isolated Rad52 protein from a higher
eukaryote. The first and second nucleic acids are complementary,
such that the Rad52 mediates annealing of the first and second
nucleic acids.
[0014] Also provided are methods of accomplishing strand exchange
comprising contacting: a) a first single stranded nucleic acid; a
double stranded nucleic acid comprising second and third single
stranded nucleic acids, wherein both the first and third nucleic
acids are complementary to the second nucleic acid; and c) isolated
Rad52 from a higher eukaryote, under conditions whereby said Rad52
mediates the annealing of the first nucleic acid to the second
nucleic acid, such that the third nucleic acid is displaced.
[0015] Methods of screening for a bioactive agent involved in
homologous recombination are also provided. The methods comprise
contacting: i) a candidate bioactive agent; ii) a first single
stranded nucleic acid; and iii) isolated Rad52 protein from a
higher eukaryote, and then screening for binding of the candidate
and the Rad52 to the nucleic acid.
[0016] Further provided are methods of screening for a bioactive
agent involved in homologous recombination comprising adding: i) a
candidate bioactive agent; ii) a first single stranded nucleic
acid; and iii) isolated Rad52 protein from a higher eukaryote, to
form a mixture. The mixture is then screened for altered biological
activity, when compared to the biological activity of the
composition in the absence of the candidate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 depicts the design of the experiment. A labeled
43-mer oligonucleotide that had been thermally annealed to
single-stranded circular M13 DNA to form a partial duplex was
displaced in the presence of HsRad52 protein by an overlapping
63-mer, called the donor strand, that had 20 extra nucleotide
residues at its 5' end. (See Panyutin et al., J. Mol. Biol., 1993).
The asteric indicates a labeled strand. The partial duplex of M13
plus a 43-mer was incubated with an excess of a 63-mer that had
been incubated with HsRad52 protein. Displacement of the
.sup.32P-labeled 43-mer was detected by agarose gel
electrophoresis.
DETAILED DESCRIPTION OF THE INVENTION
[0018] In prokaryotes, the RecA mediated homologous recombination
reaction is well-documented. This reaction requires only the
presence of the RecA protein, which binds to DNA, placing the DNA
in an extended, activated conformation which then pairs with
homologous duplex DNA and performs strand exchange resulting in
recombination. However, the mechanisms of homologous recombination
and DNA repair are not well understood in eukaryotes. In lower
eukaryotes such as yeast, a number of DNA repair mechanisms exist.
For example, in yeast, three epistasis groups of DNA damage-repair
genes have been identified (see Friedberg et al., in The Molecular
and Cellular Biology of the Yeast Saccharomyces, Broach et al.
Eds., pp 147-192, 1991 Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.). However, in higher eukaryotes, the
identification, characterization and interaction of the relevant
proteins is largely unknown.
[0019] The present invention is based on the finding that human
Rad52 will bind to nucleic acid to effect strand annealing and
strand exchange. Thus, the present invention provides compositions
comprising complexes of nucleic acids and isolated Rad52 from a
higher eukaryote.
[0020] In a preferred embodiment, the invention provides a first
single stranded nucleic acid complexed with isolated Rad52 from a
higher eukaryote. As used herein, "nucleic acid" or
"oligonucleotide" means at least two nucleotides linked together,
and may refer to either DNA or RNA, or molecules which contain both
deoxy- and ribo-nucleotides. The nucleic acids include mRNA,
genomic DNA, cDNA and oligonucleotides including sense and
anti-sense nucleic acids. "Nucleic acid" also includes nucleic acid
analogs, such as nucleic acids that contain modifications in the
ribose-phosphate backbone to increase stability and half life of
such molecules in physiological environments. For example, a
nucleic acid of the present invention will generally contain
phosphodiester bonds, although in some cases, as outlined below, a
nucleic acid analogs are included that may have alternate
backbones, comprising, for example, phosphoramide (Beaucage et al.,
Tetrahedron 49(10):1925 (1993) and references therein; Letsinger,
J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem.
81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986);
Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem.
Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141
91986)), phosphorothioate, phosphorodithioate,
O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides
and Analogues: A Practical Approach, Oxford University Press), and
peptide nucleic acid backbones and linkages (see Egholm, J. Am.
Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl.
31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al.,
Nature 380:207 (1996), all of which are incorporated by reference).
Nucleic acids containing one or more carbocyclic sugars are also
included within the definition of nucleic acids (see Jenkins et
al., Chem. Soc. Rev. (1995) pp169-176). These modifications of the
ribose-phosphate backbone may be done to increase the stability and
half-life of such molecules in physiological environments.
[0021] Unless specified, the nucleic acid may be double stranded,
single stranded, or contain portions of both double stranded or
single stranded sequence. By the term "recombinant nucleic acid"
herein is meant nucleic acid, originally formed in vitro, in
general, by the manipulation of nucleic acid by endonucleases, in a
form not normally found in nature. Thus an isolated nucleic acid,
in a linear form, or an expression vector formed in vitro by
ligating DNA molecules that are not normally joined, are both
considered recombinant for the purposes of this invention. It is
understood that once a recombinant nucleic acid is made and
reintroduced into a host cell or organism, it will replicate
non-recombinantly, i.e. using the in vivo cellular machinery of the
host cell rather than in vitro manipulations; however, such nucleic
acids, once produced recombinantly, although subsequently
replicated non-recombinantly, are still considered recombinant for
the purposes of the invention.
[0022] In a preferred embodiment, the nucleic acids of the
invention range in size from about 10 base pairs to about 100 base
pairs, with from about 20 to about 80 base pairs being preferred,
although as will be appreciated by those in the art, much longer
nucleic acids may be used as well.
[0023] The nucleic acids are complexed with isolated Rad52 protein
from a higher eukaryote. By "complexed" herein is meant that the
Rad52 protein binds to or forms a non-covalent association with the
nucleic acid. Preferably, the complex is stable to electrophoretic
conditions. In one embodiment, the complex comprises the nucleic
acid with at least one associated Rad52 protein. In a preferred
embodiment, a plurality of proteins are associated to form the
complex. Thus for example; preferred embodiments utilize ratios of
protein to nucleotide ranging from about 1:1 to about 1:10, with
from about 1:1 to about 1:5 being preferred and from about 1:1 to
about 1:2 being especially preferred. Thus, in a preferred
embodiment, the nucleic acid is substantially coated with Rad52
protein.
[0024] By "Rad52 protein" herein is meant a Rad52 protein from a
higher eukaryote. By "higher eukaryote" herein is meant a
multicellular organism, including, but not limited to, plants and
vertebrates such as birds, fish and mammals. Higher eukaryotes do
not include lower eukaryotes such as yeast and fungi. Preferred
higher eukaryotes include, but are not limited to, plants, birds,
fish and mammals, with chicken, goats, cows, rodents such as mice
and rats, salmon, and mammals being particularly preferred. Humans
are particularly preferred.
[0025] Rad52 proteins are identified as having two characteristics:
(1) Rad52 proteins have significant homology to the N-terminus of
known Rad52 proteins, including chicken, mouse and human; and (2)
Rad52 proteins will mediate annealing and strand exchange, as
described herein.
[0026] Rad52 proteins may be initially identified by homology to
other higher eukaryotic Rad52 proteins, including chicken, mouse
and human (see Park, J. Biol. Chem. 270:15467-15470 (1995); Muris
et al., Mutation Res., DNA Repair 315:295-305 (1994); Bezzubova et
al., Nucleic Acid Res. 21(25):5945-5949 (1993); Bendixen et al.,
Genomics 23:300-303 (1994); Shen et al., Genomics 25:199-206
(1995), all of which are expressly incorporated by reference). As
noted above, the N-terminus of Rad52 is highly conserved among
species; in the N-terminus, chicken Rad52 is roughly 90% homologous
to both human Rad52 and mouse Rad52, and mouse Rad52 is roughly 99%
homologous to human Rad52. Thus, proteins with significant homology
to the N-terminus of known Rad52 proteins are considered Rad52
proteins. The highly homologous N-terminus comprises from about
amino acid 36 to about amino acid 185, using the human Rad52
sequence numbering (see Muris et al., supra). All or part of this
sequence may be used to identify Rad52 proteins.
[0027] Thus, as used herein, a protein is a "Rad52 protein" if the
overall homology of the protein sequence to the N-terminal amino
acid sequences of human, chicken or mouse Rad52 is preferably
greater than about 50%, more preferably greater than about 60% and
most preferably greater than 75%. In some embodiments the homology
will be as high as about 90 to 95 or 98%. This homology will be
determined using standard techniques known in the art, such as the
Best Fit sequence program described by Devereux et al., Nucl. Acid
Res. 12:387-395 (1984) or the BLASTX program (Altschul et al., J.
Mol. Biol. 215, 403-410). The alignment may include the
introduction of gaps in the sequences to be aligned. In addition,
for sequences which contain either more or fewer amino acids than
these proteins, it is understood that the percentage of homology
will be determined based on the number of homologous amino acids in
relation to the total number of amino acids. Thus, for example,
homology of sequences shorter than those of the known Rad52
proteins, as discussed below, will be determined using the number
of amino acids in the shorter sequence.
[0028] With respect to the C-terminal sequence of Rad52, different
species show a marked lack of homology in this region, and thus
this area is not suitable for definition of a Rad52 protein. In
some embodiments, all or part of the C-terminus may be deleted,
although this area is postulated to interact with other Rad
proteins such as Rad51 and RPA, and thus may be important.
[0029] Rad5.2 proteins useful in the present invention may be
shorter or longer than the known Rad52 sequences, as long as the
protein has both sequence and functional homology to the N-terminus
of known Rad52 sequences.
[0030] In a preferred embodiment, the Rad52 protein is recombinant.
A "recombinant protein" is a protein made using recombinant
techniques, i.e. through the expression of a recombinant nucleic
acid as depicted above. A recombinant protein is distinguished from
naturally occurring protein by at least one or more
characteristics. For example, the protein may be isolated or
purified away from some or all of the proteins and compounds with
which it is normally associated in its wild type host, and thus may
be substantially pure. For example, an isolated protein is
unaccompanied by at least some of the material with which it is
normally associated in its natural state, preferably constituting
at least about 0.5%, more preferably at least about 5% by weight of
the total protein in a given sample. A substantially pure protein
comprises at least about 75% by weight of the total protein, with
at least about 80% being preferred, and at least about 90% being
particularly preferred. The definition includes the production of a
Rad52 protein from one organism in a different organism or host
cell. Alternatively, the protein may be made at a significantly
higher concentration than is normally seen, through the use of a
inducible promoter or high expression promoter, such that the
protein is made at increased concentration levels. Alternatively,
the protein may be in a form not normally found in nature, as in
the addition of an epitope tag, a purification signal such as
His.sub.6, or amino acid substitutions, insertions and deletions,
as discussed below.
[0031] Also included with the definition of Rad52 proteins are
Rad52 proteins from other organisms, which are cloned and expressed
as outlined below. Thus, probe or degenerate polymerase chain
reaction (PCR) primer sequences may be used to find other related
Rad52 proteins from other organisms. As will be appreciated by
those in the art, particularly useful probe and/or PCR primer
sequences include the unique areas of the Rad52 nucleic acid
sequence. Thus, useful probe or primer sequences may be designed to
all or part of the N-terminal sequence of the known Rad52
sequences. As is generally known in the art, preferred PCR primers
are from about 15 to about 35 U.S.C. .sctn. nucleotides in length,
with from about 20 to about 30 being preferred, and may contain
inosine as needed. The conditions for the PCR reaction are well
known in the art.
[0032] Once the Rad52 nucleic acid is identified, it can be cloned
and, if necessary, its constituent parts recombined to form the
entire Rad52 nucleic acid. Once isolated from its natural source,
e.g., contained within a plasmid or other vector or excised
therefrom as a linear nucleic acid segment, the recombinant Rad52
nucleic acid can be further used as a probe to identify and isolate
other Rad52 nucleic acids. It can also be used as a "precursor"
nucleic acid to make modified or variant Rad52 nucleic acids and
proteins as is more fully described below.
[0033] Using the nucleic acids of the present invention which
encode a Rad52 protein, a variety of expression vectors are made.
The expression vectors may be either self-replicating
extrachromosomal vectors or vectors which integrate into a host
genome. Generally, these expression vectors include transcriptional
and translational regulatory nucleic acid operably linked to the
nucleic acid encoding the Rad52 protein. "Operably linked" in this
context means that the transcriptional and translational regulatory
DNA is positioned relative to the coding sequence of the Rad52
protein in such a manner that transcription is initiated.
Generally, this will mean that the promoter and transcriptional
initiation or start sequences are positioned 5' to the Rad52
protein coding region. The transcriptional and translational
regulatory nucleic acid will generally be appropriate to the host
cell used to express the Rad52 protein; for example,
transcriptional and translational regulatory nucleic acid sequences
from Bacillus are preferably used to express the Rad52 protein in
Bacillus. Numerous types of appropriate expression vectors, and
suitable regulatory sequences are known in the art for a variety of
host cells.
[0034] In general, the transcriptional and translational regulatory
sequences may include, but are not limited to, promoter sequences,
ribosomal binding sites, transcriptional start and stop sequences,
translational start and stop sequences, and enhancer or activator
sequences. In a preferred embodiment, the regulatory sequences
include a promoter and transcriptional start and stop
sequences.
[0035] Promoter sequences encode either constitutive or inducible
promoters. The promoters may be either naturally occurring
promoters or hybrid promoters. Hybrid promoters, which combine
elements of more than one promoter, are also known in the art, and
are useful in the present invention.
[0036] In addition, the expression vector may comprise additional
elements. For example, the expression vector may have two
replication systems, thus allowing it to be maintained in two
organisms, for example in mammalian or insect cells for expression
and in a procaryotic host for cloning and amplification.
Furthermore, for integrating expression vectors, the expression
vector contains at least one sequence homologous to the host cell
genome, and preferably two homologous sequences which flank the
expression construct. The integrating vector may be directed to a
specific locus in the host cell by selecting the appropriate
homologous sequence for inclusion in the vector. Constructs for
integrating vectors are well known in the art.
[0037] In addition, in a preferred embodiment, the expression
vector contains a selectable marker gene to allow the selection of
transformed host cells. Selection genes are well known in the art
and will vary with the host cell used.
[0038] The Rad52 proteins of the present invention are produced by
culturing a host cell transformed with an expression vector
containing nucleic acid encoding a Rad52 protein, under the
appropriate conditions to induce or cause expression of the Rad52
protein. The conditions appropriate for Rad52 protein expression
will vary with the choice of the expression vector and the host
cell, and will be easily ascertained by one skilled in the art
through routine experimentation. For example, the use of
constitutive promoters in the expression vector will require
optimizing the growth and proliferation of the host cell, while the
use of an inducible promoter requires the appropriate growth
conditions for induction. In addition, in some embodiments, the
timing of the harvest is important. For example, the baculoviral
systems used in insect cell expression are lytic viruses, and thus
harvest time selection can be crucial for product yield.
[0039] Appropriate host cells include yeast, bacteria,
archebacteria, fungi, and insect and animal cells, including
mammalian cells. Of particular interest are Drosophila melangaster
cells, Saccharomyces cerevisiae and other yeasts, E coli, Bacillus
subtilis, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO,
COS, and HeLa cells, fibroblasts, Schwanoma cell lines, stem cells,
bone marrow and immortalized mammalian myeloid and lymphoid cell
lines.
[0040] In a preferred embodiment, the Rad52 proteins are expressed
in mammalian cells. Mammalian expression systems are also known in
the art. A mammalian promoter is any DNA sequence capable of
binding mammalian RNA polymerase and initiating the downstream (3')
transcription of a coding sequence for Rad52 protein into mRNA. A
promoter will have a transcription initiating region, which is
usually placed proximal to the 5' end of the coding sequence, and a
TATA box, using a located 25-30 base pairs upstream of the
transcription initiation site. The TATA box is thought to direct
RNA polymerase II to begin RNA synthesis at the correct site. A
mammalian promoter will also contain an upstream promoter element
(enhancer element), typically located within 100 to 200 base pairs
upstream of the TATA box. An upstream promoter element determines
the rate at which transcription is initiated and can act in either
orientation. Of particular use as mammalian promoters are the
promoters from mammalian viral genes, since the viral genes are
often highly expressed and have a broad host range. Examples
include the SV40 early promoter, mouse mammary tumor virus LTR
promoter, adenovirus major late promoter, herpes simplex virus
promoter, and the CMV promoter.
[0041] Typically, transcription termination and polyadenylation
sequences recognized by mammalian cells are regulatory regions
located 3' to the translation stop codon and thus, together with
the promoter elements, flank the coding sequence. The 3' terminus
of the mature mRNA is formed by site-specific post-translational
cleavage and polyadenylation. Examples of transcription terminator
and polyadenylation signals include those derived form SV40.
[0042] The methods of introducing exogenous nucleic acid into
mammalian hosts, as well as other hosts, is well known in the art,
and will vary with the host cell used. Techniques include
dextran-mediated transfection, calcium phosphate precipitation,
polybrene mediated transfection, protoplast fusion,
electroporation, encapsulation of the polynucleotide(s) in
liposomes, and direct microinjection of the DNA into nuclei.
[0043] In a preferred embodiment, Rad52 proteins are expressed in
bacterial systems. Bacterial expression systems are well known in
the art.
[0044] A suitable bacterial promoter is any nucleic acid sequence
capable of binding bacterial RNA polymerase and initiating the
downstream (3') transcription of the coding sequence of Rad52
protein into mRNA. A bacterial promoter has a transcription
initiation region which is usually placed proximal to the 5' end of
the coding sequence. This transcription initiation region typically
includes an RNA polymerase binding site and a transcription
initiation site. Sequences encoding metabolic pathway enzymes
provide particularly useful promoter sequences. Examples include
promoter sequences derived from sugar metabolizing enzymes, such as
galactose, lactose and maltose, and sequences derived from
biosynthetic enzymes such as tryptophan. Promoters from
bacteriophage may also be used and are known in the art. In
addition, synthetic promoters and hybrid promoters are also useful;
for example, the tac promoter is a hybrid of the trp and lac
promoter sequences. Furthermore, a bacterial promoter can include
naturally occurring promoters of non-bacterial origin that have the
ability to bind bacterial RNA polymerase and initiate
transcription.
[0045] In addition to a functioning promoter sequence, an efficient
ribosome binding site is desirable. In E. coli, the ribosome
binding site is called the Shine-Dalgarno (SD) sequence and
includes an initiation codon and a sequence 3-9 nucleotides in
length located 3-11 nucleotides upstream of the initiation
codon.
[0046] The expression vector may also include a signal peptide
sequence that provides for secretion of the Rad52 protein in
bacteria. The signal sequence typically encodes a signal peptide
comprised of hydrophobic amino acids which direct the secretion of
the protein from the cell, as is well known in the art. The protein
is either secreted into the growth media (gram-positive bacteria)
or into the periplasmic space, located between the inner and outer
membrane of the cell (gram-negative bacteria).
[0047] The bacterial expression vector may also include a
selectable marker gene to allow for the selection of bacterial
strains that have been transformed. Suitable selection genes
include genes which render the bacteria resistant to drugs such as
ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and
tetracycline. Selectable markers also include biosynthetic genes,
such as those in the histidine, tryptophan and leucine biosynthetic
pathways.
[0048] These components are assembled into expression vectors.
Expression vectors for bacteria are well known in the art, and
include vectors for Bacillus subtilis, E. coli, Streptococcus
cremoris, and Streptococcus lividans, among others.
[0049] The bacterial expression vectors are transformed into
bacterial host cells using techniques well known in the art, such
as calcium chloride treatment, electroporation, and others.
[0050] In one embodiment, Rad52 proteins are produced in insect
cells. Expression vectors for the transformation of insect cells,
and in particular, baculovirus-based expression vectors, are well
known in the art.
[0051] In a preferred embodiment, Rad52 protein is produced in
yeast cells. Yeast expression systems are well known in the art,
and include expression vectors for Saccharomyces cerevisiae,
Candida albicans and C. maltosa, Hansenula polymorpha,
Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P.
pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica.
Preferred promoter sequences for expression in yeast include the
inducible GAL1,10 promoter, the promoters from alcohol
dehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase,
glyceraldehyde-3-phosphate-dehydrogenase, hexokinase,
phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase,
and the acid phosphatase gene. Yeast selectable markers include
ADE2, HIS4, LEU2, TRP1, and ALG7, which confers resistance to
tunicamycin; the neomycin phosphotransferase gene, which confers
resistance to G418; and the CUP1 gene, which allows yeast to grow
in the presence of copper ions.
[0052] The Rad52 protein may also be made as a fusion protein,
using techniques well known in the art. Thus, for example, the
Rad52 protein may be made as a fusion protein to increase
expression, or for other reasons. For example, when the Rad52
protein is a Rad52 peptide, the nucleic acid encoding the peptide
may be linked to other nucleic acid for expression purposes.
[0053] Also included within the definition of Rad52 proteins of the
present invention are amino acid sequence variants. These variants
fall into one or more of three classes: substitutional, insertional
or deletional variants. These variants ordinarily are prepared by
site specific mutagenesis of nucleotides in the DNA encoding the
Rad52 protein, using cassette or PCR mutagenesis or other
techniques well known in the art, to produce DNA encoding the
variant, and thereafter expressing the DNA in recombinant cell
culture as outlined above. However, variant Rad52 protein fragments
having up to about 100-150 residues may be prepared by in vitro
synthesis using established techniques. Amino acid sequence
variants are characterized by the predetermined nature of the
variation, a feature that sets them apart from naturally occurring
allelic or interspecies variation of the Rad52 protein amino acid
sequence. The variants typically exhibit the same qualitative
biological activity as the naturally occurring analogue, although
variants can also be selected which have modified characteristics
as will be more fully outlined below.
[0054] While the site or region for introducing an amino acid
sequence variation is predetermined, the mutation per se need not
be predetermined. For example, in order to optimize the performance
of a mutation at a given site, random mutagenesis may be conducted
at the target codon or region and the expressed Rad52 variants
screened for the optimal combination of desired activity.
Techniques for making substitution mutations at predetermined sites
in DNA having a known sequence are well known, for example, M13
primer mutagenesis and PCR mutagenesis. Screening of the mutants is
done using assays of Rad52 protein activities; for example, for
binding domain mutations, competitive binding studies such as are
outlined in the Examples may be done.
[0055] Amino acid substitutions are typically of single residues;
insertions usually will be on the order of from about 1 to 20 amino
acids, although considerably larger insertions may be tolerated.
Deletions range from about 1 to about 20 residues, although in some
cases deletions may be much larger.
[0056] Substitutions, deletions, insertions or any combination
thereof may be used to arrive at a final derivative. Generally
these changes are done on a few amino acids to minimize the
alteration of the molecule. However, larger changes may be
tolerated in certain circumstances. When small alterations in the
characteristics of the Rad52 protein are desired, substitutions are
generally made in accordance with the following chart:
1 CHART I Original Residue Exemplary Substitutions Ala Ser Arg Lys
Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln
Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met,
Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu
[0057] Substantial changes in function or immunological identity
are made by selecting substitutions that are less conservative than
those shown in Chart I. For example, substitutions may be made
which more significantly affect: the structure of the polypeptide
backbone in the area of the alteration, for example the
alpha-helical or beta-sheet structure; the charge or hydrophobicity
of the molecule at the target site; or the bulk of the side chain.
The substitutions which in general are expected to produce the
greatest changes in the polypeptide's properties are those in which
(a) a hydrophilic residue, e.g. seryl or threonyl, is substituted
for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl,
phenylalanyl, valyl or alanyl; (b) a cysteine or proline is
substituted for (or by) any other residue; (c) a residue having an
electropositive side chain, e.g. lysyl, arginyl, or histidyl, is
substituted for (or by) an electronegative residue, e.g. glutamyl
or aspartyl; or (d) a residue having a bulky side chain, e.g.
phenylalanine, is substituted for (or by) one not having a side
chain, e.g. glycine.
[0058] The variants typically exhibit the same qualitative
biological activity and will elicit the same immune response as the
naturally-occurring analogue, although variants also are selected
to modify the characteristics of the Rad52 proteins as needed.
Alternatively, the variant may be designed such that the biological
activity of the Rad52 protein is altered. For example, regions
involved in DNA binding or other protein-protein interactions (such
as Rad51, etc.) may be altered.
[0059] In one embodiment, the nucleic acids and/or Rad52 proteins
of the invention are labeled. By "labeled" herein is meant that a
compound has at least one element, isotope or chemical compound
attached to enable the detection of the compound. In general,
labels fall into three classes: a) isotopic labels, which may be
radioactive or heavy isotopes; b) immune labels, which may be
antibodies or antigens; and c) colored or fluorescent dyes. The
labels may be incorporated into the complex at any position.
[0060] In a preferred embodiment, the Rad52 protein is purified or
isolated after expression. Rad52 proteins may be isolated or
purified in a variety of ways known to those skilled in the art
depending on what other components are present in the sample.
Standard purification methods include electrophoretic, molecular,
immunological and chromatographic techniques, including ion
exchange, hydrophobic, affinity, and reverse-phase HPLC
chromatography, and chromatofocusing. For example, the Rad52
protein may be purified using a standard anti-Rad52 antibody
column. If purification sequences are included, such as the
His.sub.6 tag, suitable methods are used, such as a
metal-containing column. Ultrafiltration and diafiltration
techniques, in conjunction with protein concentration, are also
useful. For general guidance in suitable purification techniques,
see Scopes, R., Protein Purification, Springer-Verlag, NY
(1982).
[0061] Once expressed and purified if necessary, the Rad52 proteins
are complexed with the desired nucleic acid. Generally, this is
done by adding the isolated Rad52 proteins with nucleic acids, as
outlined herein. As outlined above, the amount of Rad52 protein
added to the nucleic acid can vary. Preferred embodiments utilize a
1:1 ratio of protein to nucleotide within the nucleic acid; i.e. 10
molecules of Rad52 for a nucleic acid 10 nucleotides in length.
[0062] Binding of Rad52 to nucleic acid may be verified by gel
migration studies, as will be appreciated by those in the art and
outlined in the Examples.
[0063] It should be noted that the mechanism of binding of Rad52
protein to nucleic acid is both ATP and Mg+2 independent. That is,
unlike RecA or Rad51 binding to nucleic acid, Rad52 protein is able
to bind to nucleic acid in the absence of co-factors or specific
ions.
[0064] In a preferred embodiment, the isolated Rad52 protein is
complexed to a first single stranded nucleic acid, as outlined
herein. This complex is capable of mediating the annealing of this
first single stranded nucleic acid to a complementary single
stranded nucleic acid. Thus Rad52 proteins are defined functionally
as well. By "mediating" herein is meant that the presence of the
Rad52 protein will either (1) allow annealing of two pieces of
nucleic acid which in the absence of the Rad52 would not
spontaneously anneal under the reaction conditions; or (2) will
increase the rate at which annealing occurs. Thus, the Rad52
protein/nucleic acid complex is recombination active, i.e. able to
anneal, or do strand exchange, or both.
[0065] Thus, for example, it has been shown that the presence of
some types and number of mismatches as between two single stranded
nucleic acids will prevent spontaneous annealing (see for example
Panyutin et al., J. Mol. Biol. 230:413-424 (1993), hereby expressly
incorporated by reference). Similarly, the presence of certain
buffer conditions, even in the absence of mismatches, will prevent
or reduce spontaneous annealing (see the Examples). Thus, in the
presence of Rad52, the rate at which the single stranded nucleic
acids will anneal is faster than the rate (if any) in the absence
of Rad52.
[0066] Annealing is done with complementary nucleic acids. By
"complementary" herein is meant one of two things. In a preferred
embodiment, the two single stranded nucleic acids are sufficiently
complementary to hybridize or anneal under at least low stringency
conditions, with complementarity preferably being sufficient to
allow hybridization under moderate or high stringency conditions.
Generally, low, moderate and high stringency conditions are known
in the art. Suitable representative low, moderate and high
stringency conditions are known in the art.
[0067] Additionally or alternatively, complementary means that the
two single stranded nucleic acids are at least sufficiently
complementary to be capable of base pairing in the presence of
Rad52. That is, since Rad52 mediates the annealing of single
stranded nucleic acids that would not ordinarily anneal, nucleic
acids which do not spontaneously anneal or do so only very slowly,
such that they would not normally be considered "complementary" are
considered complementary for the purposes of this application if
they will anneal in the presence of Rad52 protein.
[0068] Thus, complementary nucleic acids may be: perfectly
complementary, i.e. contain no mismatches; significantly
complementary, i.e. contain from less than about 1% to about 10%
mismatches; or minimally complementary, i.e. contain roughly about
10 to 30 or 40% mismatches, but still be sufficiently complementary
to allow annealing in the presence of Rad52 protein.
[0069] The compositions of the invention may further comprise a
second single stranded nucleic acid complexed to Rad52 protein. In
a preferred embodiment, this second single stranded nucleic acid is
complementary, as defined above, to the first single stranded
nucleic acid with bound Rad52.
[0070] The compositions of the present invention may comprise other
elements, such as other proteins, or specific binding moieties for
purification or assay purposes.
[0071] In a preferred embodiment, the invention provides methods of
accomplishing strand exchange. In this embodiment, the compositions
of the invention comprise a first single stranded nucleic acid
complexed with Rad52, and a double stranded nucleic acid comprising
a second and a third single stranded nucleic acid. In this
embodiment, the first and third single stranded nucleic acids are
complementary to the second nucleic acid, such that strand exchange
between the third and first nucleic acids may occur. Thus, the
third nucleic acid is displaced from the double stranded nucleic
acid and replaced by the first nucleic acid, with annealing of the
first and second nucleic acids, to provide a double stranded
nucleic acid comprising the first and second nucleic acids, leaving
the third nucleic acid as a single stranded nucleic acid.
[0072] The invention also provides methods of making double
stranded nucleic acid comprising contacting a first single stranded
nucleic acid, a second single stranded nucleic acid, and isolated
Rad52 protein from a higher eukaryote. This contacting may be done
in any order. For example, in a preferred embodiment, the first or
the second nucleic acid and the Rad52 proteins may be contacted
first, forming a nucleic acid-Rad52 complex, followed by addition
of the other nucleic acid, which leads to annealing of the first
and second nucleic acids to form a double stranded nucleic acid.
Alternatively, the first and second nucleic acids may be added,
followed by the addition of the Rad52 protein. Assays to measure
whether annealing has been accomplished are well known in the
art.
[0073] In a preferred embodiment, the compositions of the invention
are used in screening assays for bioactive agents involved in
homologous recombination. In this embodiment, candidate bioactive
agents are added to the compositions of the invention. By
"candidate bioactive agent" herein is meant any molecule, e.g.
proteins, including antibodies, oligopeptides, small organic
molecule, polysaccharide, polynucleotide, etc., which may be tested
for the ability to alter the bioactivity of the composition.
Particularly preferred candidate bioactive agents are proteins, and
particularly preferred are other Rad52 epistasis group
proteins.
[0074] In one embodiment, the method consists of adding a candidate
bioactive agent, a first single stranded nucleic acid, and isolated
Rad52 protein, to form a mixture. The addition may be done in any
order. In a preferred embodiment, the first nucleic acid and the
Rad52 protein are complexed prior to the addition of the candidate
agent. Alternatively, the components are added simultaneously, or
the candidate agent is added first to either the first nucleic acid
or the Rad52 protein. The mixture is then assayed for altered
biological activity.
[0075] By "bioactivity" or "biological activity" herein is meant
any of a number of potential biological activities of the
compositions of the invention. Biological activity includes, but is
not limited to, nucleic acid binding, annealing, strand-exchange,
and homology scanning. Generally, the biological activity will be
tested using well known procedures. By "altered biological
activity" herein is meant that the biological activity of the
mixture of the candidate bioactive agent and the Rad52-protein
complex is different, i.e. increased or decreased, relative to the
biological activity in the absence of the candidate agent.
[0076] In one embodiment the desired bioactivity is binding to the
complex of Rad52 and nucleic acid. Thus, in this embodiment, the
screening assay is a binding assay; that is, the desired
bioactivity is binding to nucleic acid, either in the presence of
Rad52, i.e. binding to the Rad52-nucleic acid complex, or to
prevent Rad52 binding. In this embodiment, one or more of the
molecules may be joined to a label, where the label can directly or
indirectly provide a detectable signal. Alternatively, the
determination of binding activity is done via gel mobility
studies.
[0077] The following examples serve to more fully describe the
manner of using the above-described invention, as well as to set
forth the best modes contemplated for carrying out various aspects
of the invention. It is understood that these examples in no way
serve to limit the true scope of this invention, but rather are
presented for illustrative purposes. All references cited herein
are incorporated by reference.
EXAMPLES
[0078] Materials. T4 phage DNA ligase and restriction enzyme (Not
I) were obtained from New England Biolabs, MA, USA. AmpliTaq
ploymerase was from Perkin-Elmer, Conn., USA. Expand High Fidelity
PCR Kit was from Boehringer Mannheim Co, Ind., USA. Expression
vector pQE-31, E. coli M15 (pREP4) strain, Probond nickel matrix
was obtained from Invitrogen, CA, USA.
[0079] Cloning of the HsRad52 gene. The whole coding sequence of
human Rad52 (HsRad52) was amplified by PCR from human thymus cDNA
library. Sequences of the upstream and downstream primers are
EG182: CGCGGATCCGATGTCT GGGACTGAGGAAGCAA and EG225:
GTAGGATCCTGAGCCTCAGTTAAG ATGG. Underlined sequences are homologous
to the published 5' end and 3' end sequence of the HsRad52 gene
(10, 12). For PCR, 0.5 .mu.g of DNA was used in mixture containing
each primer at 200 nM, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1 mM
MgCl2, 0.01% gelatin, each of the four dNTP's at 200 .mu.M and 2.5
units of AmpliTaq ploymerase. The reaction mixture was heated at
95.degree. C. for 3 min and used in a PCR consisting of 30 cycles
of 94.degree. C. for 1 min, 60.degree. C. for 1 min, and 72.degree.
C. for 1 min. The resulting DNA fragment was labeled with [-32P]
dCTP by random priming (24) and used as a probe for isolation of a
HsRad52 clone from human testis cDNA library in lambda Charon BS
phage (25). DNA isolated from a hybridization-positive clone was
digested by Not I and treated with T4 DNA ligase. The ligation
mixture was used for transformation of E. coli SY204. Ampicilin
resistant transformants contained a plasmid, designated pEG970,
which carries HsRad52 cDNA. The plasmid was used as a template for
amplification of the coding region of HsRad52 gene by PCR reaction.
The reaction was carried out by using Expand High Fidelity PCR Kit
and primers EG182 and EG225. The resulting DNA fragment was
inserted into expression vector pQE-31 in frame with 5' end
sequence coding for a series of six histidine residues that
function as a metal-binding domain in the translated fusion
protein.
[0080] Isolation of HsRad52 protein. Plasmid pEG2 which carries the
whole coding sequence of the HsRad52gene in the vector pQE-31 was
introduced into E. coli M15 (pREP4). Synthesis of the protein is
under the control of the E. coli phage T5 promoter and two lac
operator sequences and can be induced by J3-D-thiogalactopyranoside
00(IPTG). For the isolation of HsRad52 protein, E. coli M15
(pREP4)/pEG2 cells were grown in LB medium at 37.degree. C. to
OD590=0.6 and induced with 2 mM IPTG. Cells were harvested 2 hrs
after induction and resuspended in buffer containing 50 mM Tris-HCl
(pH8.0), 10% sucrose. Cells were lysed by the addition 10 mg/ml of
lysozyme. Solution was kept on ice and stirred for 30 min, then 350
mM KCl and 0.5% Brij 58 were added and stirred on ice for another
60 min. Cell debris was spun down by centrifugation at 35000 rpm
for 75 min at 4.degree. C. HsRad52 protein from the supernatant was
loaded directly on Probond nickel column, that was equilibrated
with 50 mM Tris-HCl (pH 8.0), 300 mM NaCl. The column was washed
with 20 volumes of washing buffer (50 mM Tris-HCl (pH 6.0), 300 mM
NaCl, 50 mM imidazole) and the bound protein was eluted with 50 mM
Tris-HCl (pH 6.0), 300 mM NaCl and 500 mM imidazole. Peak fractions
were dialyzed against 50 mM Tris-HCl (pH 8.0), 200 mM NaCl and 10%
glycerol. The purified protein was near homogeneous (FIG. 1) and
did not contain any detectable nucleases under the reaction
conditions.
[0081] Binding of HsRad52 protein to single-stranded and
double-stranded DNA. The DNA binding activity of HsRad52 protein
was determined by band shift assay. The reaction mixtures (10
.mu.L) contained 20 mM Tris-HCl (pH 8.0), 1 mM MgCl2, 75 mM NaCl,
10 mM .beta.-mercatoethanol, 1 .mu.M DNA and 1M HsRad52 protein.
The reaction mixtures with or without HsRad52 protein were
incubated at 37.degree. C. for 30 min and were electrophoresed on
0.8% agarose gel. The gels were subsequently dried and examined by
autoradiography. Single-stranded DNA used in this experiment was a
43-mer oligonucleotide, end labeled with 32-g ATP using
polynucleotide kinase. Blunt ended double-stranded was obtained by
thermally annealing a complementary 43 to the labeled 43-mer
oligonucleotide. Similarly, partial duplexes with 3' tail or 5'
tail were obtained by thermally annealing the labeled 43-mer with a
complementary 63-mer olgonucleotide which has an additional 20
nucleotides at the 3' end or 5' end. HsRad52 protein bound equally
efficiently to single-stranded DNA, double-stranded DNA and partial
duplexes with 3' or 5' overhanging tails (data not shown). All of
the DNA was bound by HsRad52 protein and the resulting protein-DNA
complex moved slowly in 0.8% agarose gel (data not shown). The
binding of HsRad52 protein was optimal at 1:1 ratio of protein to
nucleotide concentrations. At lower concentrations of protein (one
protein for every two or four nucleotides) binding was incomplete
and resulted in a band that was smeared (data not shown).
[0082] In order to detect the ability of HsRad52 protein to anneal
complementary strands of DNA and also to assess its role in branch
migration/strand exchange, we used an assay described by Panyutin
and Hsieh, supra.
[0083] HsRad52 promotes annealing. The design of the experiments is
shown in FIG. 1.
[0084] Reactions were carried out with a partial duplex made by
annealing 32P-labeled oligonucleotide 43-mer to M13, a 12.5-fold
molar excess of donor oligonucleotide with respect to the number of
molecules of M13 single strands and a molar ratio of 1:1.5 of
HsRad52 protein with respect to the nucleotide residues of the
donor oligonucleotide. Reaction mixtures (10 .mu.L) contained 20 mM
Tris-HCl at pH 8.0, 20 mM Nacl, 10 mM B-mercapto ethanol 0.6 .mu.M
of 63-mer and 0.4 .mu.M of HsRad52. Reaction mixtures were
preincubated at 37.degree. C. for 15 min followed by the addition
of 5 .mu.M of M13/43-mer partial duplex and the reactions were
incubated at 37.degree. C. for a further 60 min. After the
incubation reactions mixtures were deproteinized with the addition
of 0.1 mg/mL proteinase K, 0.5% SDS at 37.degree. C. for 15 min.
Samples were analyzed by gel electrophoresis on 0.8% agarose gels,
and quantitated by phosphorimaging. Conditions for all the control
reactions were the same as with HsRad52 protein.
[0085] A 43-mer oligonucleotide radiolabeled at its 5' end was
annealed to single-stranded circular M13 DNA to form a partial
duplex (called as M13/43-mer duplex) Displacement of the labeled
43-mer by a longer, overlapping, unlabeled 63-mer (called as donor
strand) requires first the the donor oligonucleotide anneal to one
side of the partial duplex region, followed by a strand exchange
reaction. Depending on conditions, annealing, strand exchange, or
both may occur spontaneously or by a catalyzed reaction. The
uncatalyzed reaction is usually called as branch migration.
[0086] Using such substrates, Panyutin and Hsieh (supra) previously
studied spontaneous branch migration in reaction mixtures that
contained 50 mM Tris-HCl (pH 7.5), 50 mM NaCl and a 250-fold excess
of the donor oligonucleotide. They found that 4 mismatches largely
blocked the uncatalyzed reaction. Alternatively, spontaneous branch
migration is reduced significantly, even in the absence of
mismatches, when the reaction conditions are as follows: 20 mM
Tris-HCl (pH 7.5 or 8.0), 20 mM Nacl and a 25-fold molar excess of
the overlapping single-stranded oligonucleotide. These conditions
were used for studying the action of HsRad52 protein.
[0087] The donor oligonucleotide was incubated with HsRad52 under
the conditions just described for 10 min and added the partial
duplex to start the strand displacement reaction. HsRad52 promoted
the displacement of the 43-mer oligonucleotide from the M13/43-mer
duplex by a fully homologous 63-mer that had 20 extra nucleotide
residues at its 5' end as determined on gels. Displacement was
minimal in the absence of HsRad52, did not occur in the absence of
63-mer oligonucleotide, or when the oligonucleotide was
heterologous. Displacement also did not occur when the
oligonucleotide was homologous only at its overhanging 5' end,
indicating that displacement depends upon replacement of one
homologous strand by another, rather than on a helicase activity of
HsRad52 protein. However, presence of 4 mismatches in the 63-mer
oligonucleotide reduced the strand displacement to roughly about
10%. The efficiency of this reaction could be increased to about
24% by increasng the concentration of the invading oligonucleotide
to 50-fold molar excess (data not shown).
[0088] Displacement of the 43-mer oligonucleotide from the partial
duplex occured even when the order of addition was reversed i.e.
when the HsRad52 was first incubated with the partial duplex and
the donor was added later to start the reaction (data not shown).
Sequence CWU 1
1
2 1 32 DNA Artificial sequence EG182 primer 1 cgcggatccg atgtctggga
ctgaggaagc aa 32 2 28 DNA Artificial sequence EG225 primer 2
gtaggatcct gagcctcagt taagatgg 28
* * * * *