Download Characterization of the Human Homologue of

(CANCER RESEARCH57. 2378-2383. June 15. 19971
Advances in Brief
Characterization of the Human Homologue of RAD54: A Gene Located on
Chromosome 1p32 at a Region of High Loss of Heterozygosity
in Breast Tumors'
Debora Rasio,2 Yoshiki Murakumo,2 David Robbins, Tim Roth, Aaron Silver, Massimo Negrini, Carl Schmidt,
John Burczak, Richard Fishel,3 and Carlo M. Croce
Kimmel Cancer institute and Kimmel Cancer Center. Thomas Jefferson University, Philadelphia.
SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406 (C. S.. J. B.. D. R.)
Abstract
A search of the Human Genome Sciences database of expressed se
quence-tagged DNA fragments, for sequences containing homologj@to
known yeast DNA recombination and repair genes, yielded a eDNA
fragment with high homologj@to RADS4. Here we describe the complete
eDNA sequence and the characterization of the genomic locus coding for
the human homologue of the yeast RA.D54 gene (hR4D54). The yeast
RAD54
belongs
to the R.4D52
epistasis
group
and appears
to be involved
in both DNA recombination and repair. The hRAD54 gene maps to
chromosome lp32 in a region of frequent loss of heterozygosity in breast
tumors and encodes a protein of Mr 93,000 that displays 52% identity to
the yeast RADS4 protein. The hRADS4 protein sequence additionally
contains all seven of the consensus segments of a superfamily of proteins
with presumed or proven DNA helicase activity. Mutations in genes with
consensus helicase homology have been found in cancer-prone syndromes
such as xeroderma pigmentosum and Bloom syndrome as well as Wern
er's syndrome, in which patients age prematurely, and the X-linked
mental
retardation
with
a-thalassemia
syndrome,
ATR-X.
We have
ex
amined the hR4D54 gene in several breast tumors and breast tumor cell
lines and, although the gene region appears to be deleted in several
tumors, at present we have found no coding sequence mutations.
Introduction
A loss of DNA repair functions that results in elevated mutation
rates has been proposed to be the driving force responsible for the
multiple mutations found in the development of tumors (1). The
foundation for this hypothesis was garnered when it was shown that
mutations of the human mismatch repair genes hMSH2 and hMLHJ
accounted for the majority of hereditary nonpolyposis colon cancer
cases (2—7),a common cancer predisposition syndrome that accounts
for 6—10%of colon cancers (8—10).Although alteration of mismatch
repair functions has been found to increase the rate of mutation in
bacteria, yeast, and human cells, presumably via misrepair of misin
corporation errors during chromosome replication (1 1—13),there ap
peared to be a number of other mechanisms in which altered repair
functions could lead to higher mutation rates.
One of the most common alterations observed in tumors is the loss
or rearrangement of chromosomes (14—16).The mechanism of this
type of alteration has been proposed to be the result of chromosome
pairing disorders prior to mitotic dysjunction, aberrant recombina
Received 10/18/96; accepted 5/12/97.
The costs of publication of this article were defrayed in part by the payment of page
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18 U.S.C. Section 1734 solely to indicate this fact.
I This
work
was
supported
by Outstanding
Investigator
Grant
in accordance with
CA39860
(to C. M.
3 To
whom
requests
for
equally
reprints
be
addressed,
(hRAD54)
and mapped
it to chromosomal
region
ip32. The short arm
of chromosome 1 shows a complex pauern of internal deletions (26),
suggesting the presence of tumor suppressor genes required for nor
mal cellular maintenance. For example, chromosomal band lp32 has
been identified as one of the four minimal regions of chromosome 1
deletion in breast carcinomas (27—29),and tumors of neural crest
origin like medullary thyroid carcinomas and pheochromocytomas,
together with meningiomas, show loss of genetic material (LOH4) at
1p32—pter(30—34).The predicted gene product ofthe hRAD54 is a Mr
93,000 protein belonging to a superfamily of DNA helicases (35).
Mutations in genes with DNA helicase function have been found to be
responsible for cancer-prone syndromes like xeroderma pigmentosum
(36—38)and Bloom syndrome (39) as well as Werner's syndrome
(40), in which patients age prematurely, and the X-linked mental
retardation with a-thalassemia syndrome, ATR-X (41, 42). These
results suggested that the hRAD54 gene could be a candidate modifier
gene in tumors that display allelic imbalance at lp32. To test our
hypothesis, we have determined the intron/exon structure of the
hRAD54 gene and performed SSCP analysis on tumor genomic DNA
to detect any potential mutations.
Materials
and Methods
Database
Search
sequence
from
accession
number
for Novel DNA Repair
the yeast
DNA
M63232)
computer
excision
Enzymes.
repair
was used to screen
enzyme
The amino acid
RAD54
(GenBank
the HGS computer
database
software designed by the Genetics Computer
Group (University of Wisconsin, Madison, WI). The HGS database contains
to this work.
should
19107 (D. R.. Y. M.. T. R., A. S.. M. N.. R. F.. C'. M. C'.). and
tional repair processes, or loss of cell cycle check point functions that
normally maintain chromosomal integrity (17, 18). The study of lower
eukaryotes has identified several genes involved in chromosome
pairing and recombinational repair which, if conserved in human
cells, might be candidates for alteration in developing tumors (re
viewed in Ref. 19).
Yeast cells with mutations in the RAD54 gene display severe
defects in the repair of DNA damage induced by ionizing radiation
(20), in spontaneous and induced mitotic recombination (21), and in
HO-catalyzed mating-type interconversion (22, 23). The RAD54 gene
belongs to the RAD52 epistasis group that additionally includes
RAD5I, RAD52, RAD55, and RAD57 (20, 24).
Genes from this group have an established role in the repair of
DNA double strand breaks, which are lethal if unrepaired in haploid
yeast cells (25). A postulated role for the yeast RAD54 gene in
recombinational repair is to provide access to regions of chromatin,
thus making them available for recombination (24).
We have cloned the human homologue of the RAD54 gene
with the TFASTA
C.)
and NIH Grants CA56542 and CA57007 (to R. F.).
2 These authors contributed
Pennsylvania
at
Kimmel
Cancer
Institute
and Kimmel Cancer Center, Thomas Jefferson University, 233 South 10th Street,
Philadelphia,
PA 19107. Phone: (215) 503-1345; Fax: (215) 923-1098; E-mail:
[email protected].
4 The
abbreviations
used
are:
LOH,
loss
of
heterozygosity;
SSCP,
single-strand
con
formational polymorphism; HGS, Human Genome Sciences; EST, expressed sequence
tag; RACE, rapid amplification of cDNA ends; ORF, open reading frame.
2378
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HUMAN HOMOLOGUE OF RAD54
nucleotide sequence information of ESTs (43), which identify a diverse col
for 20 5, 72°C for 30 s, followed
lection of cDNAs derived from more than 400 cDNA libraries. One EST
(designated C2) was found to have significant homology but not identity to the
were electrophoresed on a 1.5% agarose gel and visualized by ethidium
bromide staining. The result of the screening was analyzed with the statistical
RAD54proteinsequence,62% identityin a 126-aminoacid overlap.At the
program
DNA sequence level, the EST was found to have a 51% identity to the yeast
RAD54 over 492 bases. In a separate comparison, the amino acid sequence
from the human DNA excision repair enzyme ERCC6 (GenBank accession
number L0479l; Ref. 44) was also used to screen the HGS database with
score, >3.0), was given.
Isolation of Genomic Clones. PrimersC2f and C2r were used for PCR
screening
revealed
that one of them contained
a 3.2-kb
Northern
Northern
Analysis.
A probe
the entire hRAD54
blot (Clontech)
derived
from the insert
of the cDNA
ORF was used to hybridize
according
to the manufacturer's
sensus
and thymus
with much
lower
expression
in small
intestine
on chromosome
BAC library (Research
Genetics).
lp32 (LOD
Four positive
at sites where
from the hRAD54
was used for the comparison
cDNA
the sequence
sequence.
of the sequences.
of the genomic
The computer
Intron
program
product
FASTA
size was determined
by
direct sequencing or by gel electrophoresis of inter-exon PCR products. A
5-untranslated region upstream (1.5 kb) of the hRAD54 start site was se
quenced additionally.
Analysis of the 5 ‘-untranslated region for transcription
factor binding motifs was performed using the GENETEX-MAC analysis
program.
SSCP Analysis. Genomic DNAs from 17 breast cancer cell lines and 20
sporadic
breast tumors
vidual exons
were analyzed
were designed
for mutations.
on the basis of intronic
Primers
sequences.
amplifying
mdi
PCR reactions
were performed in 10 @.d
of final volume with 20 ng of genomic DNA using
0.5 units of Taq DNA polymerase, 200 @.tM
each of dATP, dGTP, and dTI'P,
2.0 @.LM
ofdCTP,
and [a-32P]dCTP
at 0.5 MCi/reaction.
Cycles
were as follows:
95°Cfor 5 mm; then 22 cycles of 94°Cfor 15 s; 57°Cfor 20 s; 72°Cfor 20 s;
followed by 5 mm of extension at 72°C.SSCP products were run on a 0.5 x
clone
MDE gel (FMC BioProducts) for 16 h and exposed for visualization by
autoradiography
The filter
was washed with 0.5X SSC-l% SDS for 1.5 h at 60°Cand subjected to
autoradiography for 16 h. A highly expressed 3.2-kb transcript was detected in
testis
splice junctions
differed
a multitissue
protocol.
genomic
D1S443,
Determination of Exon-Intron Boundaries and Intron Sizes. Primers
derived from the cDNA were used for bidirectional sequencing of the genomic
BAC clones. Exon-intron boundaries were identified by the presence of con
insert encoding
the entire ORF. Double-strand sequencing of this clone was performed by a
cycle sequencing program using the dye-deoxynucleotide kit and Taq DNA
polymerase (Perkin-Elmer). Nucleotide sequences were determined by an
automated Applied Biosystem Sequencer model 377.
containing
of a human
to marker
Qiagen Plasmid Midi kit and used as template for sequencing analysis.
identity in an 84-amino acid overlap). At the DNA sequence level, the EST
was found to have a 64% identity to the human ERCC6 over 220 bases. The
EST sequence was compared to the HGS database using the FASTA program
(Genetics Computer Group), and two additional ESTs were found to give
overlapping sequence identity. The three ESTs were assembled into a single
consensus sequence of 401 bases with the SEQMAN routine of the LaserGene
software package (DNAstar).
eDNA Cloning. Primersfrom the 5' and 3' end of the C2 EST were used
on peripheral blood cDNA to generate a PCR-derived probe for the screening
of a normal testis ADR2 cDNA library (Clontech) by conventional plaque
hybridization. Eight clones were initially isolated and excised in pDR2 plasmid
according to the manufacturer's instructions. Sequence analysis of the eight
clones
and linkage
at 72°C. PCR products
clones were identified, and DNA from two of them was purified with the
TFASTA. The same EST homologous to yeast RAD54 was found to have
significant homology but not identity to the ERCC6 protein sequence (36.9%
positive
RHMAP,
by 5 mm of extension
on KODAK
X-AR5
film.
Results
and colon
Search of the HGS database for ESTs having homology to known
yeast repair genes revealed the presence of a 400-bp EST, C2, having
62% homology to the yeast RAD54 protein. PCR primers were
prepared
from human testis poly(A)
mRNA (Clontech)
using a Promega
designed that specifically amplified a l20-bp product when the human
cDNA synthesis kit. The cDNA was then treated with RNase H, extracted with genomic DNA was used as a template and not when hamster DNA
phenol, and precipitated with ethanol. The single-strand cDNA was used was used. This primer pair was used to screen the GeneBridge 4
directly in a RACE reaction. Four different oligonucleotides were used in this
radiation hybrid panel constructed in hamster recipient cells to deter
experiment: RACE anchor, 5'-CA COG ATC CAC TAT CGA UC TGG
AAC; RACE primer, 5'-CCA GAA TCG ATA GTG GAT CCG T; mine the chromosomal map position of C2. The primers amplified a
band of the expected size in 13 of the 93 hybrids forming the panel,
hRAD54-A, 5'-GCC AAG CTC CTC CTC ATC CT; and hRAD54-B, 5'-CTG
Ccc TCA GGT‘ITf
CrC TTG. Di-deoxythymine(Perkin-Elmer)was added and computational analysis of the screening linked the C2 EST to
to the RACE anchor using terminal transferase (Pharmacia Biotech). The marker D1S443, located on chromosomal band lp32. The screening
RACE anchor was then ligated to the single-stranded cDNA for 6 h at 37°C was repeated twice, yielding identical results. To clone the full-length
using RNA ligase (New England Biolabs) in 50 mM Tris (pH 7.8), 10 mM
cDNA, we synthesized primers that amplified the entire C2 EST to
magnesium chloride, 1 mMhexamine cobalt chloride, 20 ,.LMAlP, 10 @g/ml generate a PCR probe that was used for plaque hybridization of a
BSA, and 50% polyethyleneglycol 8000, and this cDNA was used directly for testis cDNA library. Upon screening, eight different A clones, B I to
PCR. The first round of PCR was carried out for 35 cycles using the RACE B8, were isolated. The cDNA inserts were excised from the A clones
primer and hRAD54-A primer. One @.d
of the resulting PCR product was then
to generate plasmid clones, and nucleotide analysis was performed on
used as a template for a second round of PCR using the RACE primer and
both strands using insert-derived primers and primers derived from
hRAD54-B primer for 25 cycles. PCR reaction was performed in 75 mMTris
9.0, 20 mMammonium sulfate, 2 mMmagnesium chloride, 0.01% Tween 20, the newly generated sequences. Sequence analysis of plasmid clone
10% glycerol, 0.2 mMeach of the 4 deoxynucleotide triphosphates, and 0.4 @M B5 (pBS)showedthatitsinsert,containing3135 nucleotides,included
of each primer. Cycling temperatures consisted of 30 s at 94°C,1 mm at 50°C, an entire ORF with substantial homology to the yeast RAD54 protein
and 1 rain at 72°Cand was performed in a Perkin-Elmer 2400. Amplified (termed hRAD54). Nucleotide sequence of the hRAD54 cDNA and its
fragments were cloned using a TA cloning kit (InVitrogen) and sequenced predicted ORF are shown in Fig. 1. The first in-frame ATG start
using a Sequenase 3.0 sequencing kit (United States Biochemical) or a ABI373 codon is present at nucleotide position 675, upstream of the ATG, in
sequencer. The sequences 5‘-RACE
product was found to be identical to the the 5'-untranslated region; in-frame stop codons are present at posi
cDNA clone, suggesting that we have isolated the full-length hRAD54 cDNA. tions 657, 627, and 564. Downstream of the ATG, the ORF extends
At present, we have been unable to identify or sequence the 4.0-kb mRNA until nucleotide 2915. A potential consensus AATAAA polyadenyl
observed by Northern analysis.
ation signal sequence is found at position 3090 of the cDNA, which
Chromosomal Mapping. Primers C2f (AGCCCTGAC1TFGTCUCA)
upstream
of the poly(A)
tail. The predicted
protein
and C2r (GCTTGTTCATCCATFGGCT), derived from C2 EST, were able to is 20 nucleotides
of the hRAD54 gene contains 747 amino acids, encoding a Mr 93,000
amplify a l20-bp product on human genomic DNA. They were used for PCR
screening of the GeneBridge 4 radiation hybrid mapping panel (Research protein with 52% identity to the yeast RAD54 protein.
To isolate genomic clones containing the hRAD54 gene, a human
Genetics). PCR reactions were carried out in a lO-pi final volume with the
following conditions: 95°Cfor 5 mm, then 35 cycles of 94°Cfor 15 s, 57°C BAC library was screened by PCR, and two positive clones were
mucosa. An approximately 4.0-kb minor transcript was also observed in testis.
5'-RACE Amplification of hRAD54. 5'-RACE (Apte, 1993 #842) was
used to identify the 5' end of the hRAD54 cDNA. Random primed cDNA was
2379
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HUMAN HOMOLOGUE OF R4D54
1
GGTCTTGGCGGGTCGGTGAGTCTTGGCGGCTGTTAACGCGCGCTTTGGGAACAGGAAGGTTGAGA
66 GAGAGGTGCTGGGGTCTGCGTCTATCTCTGTCGCTCTTTTCAGCCCCTCCTGGTATTCCCCTCCTAACCTGGGTTTTTTACACGCCC
15 3 GCGTGGCTTCCTGCTCGACCTCCCTGAGTCTGATCCTGGTTTCCACCTCCAGCCCTGGGAAATTTCCTTTCTCCAGACTCGCCCTCC
2 40
CCACCCGGGCCTCGGACTTTCACCCCAGCTTCTCTCTCCTGGCCAGTGATTACCCACCCCCAATCCCACCCCGCCCCGCCGCGCAAC
327 TACCTCCTCCCTTCACCCGGACTGGGACCATCATCCCCACTCCACTCCGCCCAGTCTGGGACTCCACCTGCCTCCTCcCCAATCCCA
4 14 CACTAATCTCTGCTTGGTCTCTTCCTCTTTGGCCTAATCTCTCGTCTCGGCTTATTGGGGACGGCCACTCTCACAGTTTGGTTCCAA
50 1 ACACCAGTTCCTGGATGGATTCCCGCCATCCATGCCCCCTCTTTAATTAGCCGGTCCTCTCAATAATGTAGCAGCCCCCTCTACAGA
588
TTAGACCCTGGTCCTACACTCTTAGCCGCTGCCTGCTTTTGACCTTTGGCTCATGGGTACTTGACGTTTTAAACTCCTAGGCCCAGG
Met Arg Arg Ser Leu Ala Pro 6cr Gin Leu Ala Lye Arg Lye Pro Glu Gly Arg Ser Cys Asp Aep
675 ATG AGG AGG AGC TTG GCT CCC AGC CAG CTG GCC AAG AGA AAA CCT GAA GGC AGG TCC TGT GAT GAT
Glu Asp
Trp
Gb
Pro Gly Leu Val Thr
Pro Arg
Lye Arg Lys 6cr Ser Ser Glu Thr Glu
Xl. Gln
22
44
742 GAA GAC TGG CAA CCT GGC CTA GTG ACT CCT AGG AAA CGG AAA TCC AGC AGT GAG ACC CAG ATC CAG
Glu Cys Pbs Lsu Ser Pro Phe Arg Lys Pro Leu Ssr Gin Leu Thr Am
Gin Pro Pro Cys Leu Asp
808 GAG TGT TTC CTG TCT CCT TTT CGG AAA CCT TTG AGT CAG CTA ACC AAT CAA CCA CCT TGT CTG GAC
S.r Ser Gin His Giu Ala Phe Ii. Arg Ser lie L.u Ser Lys Pro Ph. Lys Val Pro Ii. Pro Asn
874 AGC AGT CAG CAT GAA GCA TTT ATT CGA AGC ATT TTG TCA AAG CCT TTC [email protected] CCC ATT CCA AAT
Tyr Gin Gly Pro Leu Gly Ser Arg Ala Leu Giy L.u Lys Arg Ala Giy Vai Arg Arg Ala Leu His
9 40 TAT
Asp
1006 GAC
Lys
1072 AAG
Pro
1138 CCT
His
1204 CAT
Thr
1270 ACA
Ser
1 3 36 AGC
Ii.
1402 ATC
Arg
1468 AGG
Lye
1534 AAA
Tyr
1600 TAC
Asp
1666 GAT
Phe
CAA
Pro
CCC
Leu
CTT
His
CAT
Giy
GGC
Leu
CTT
Leu
CTG
Asp
GAT
Val
GTG
Giy
GGA
Gin
CAA
Lsu
CTG
Lye
GGT
Leu
CTG
Asp
GAC
Gin
CAG
Cys
TGC
Leu
TTA
Val
GTG
Giy
GGA
Her
TCT
Ser
AGT
Ala
GCC
L.u
CTT
Lye
CCT
Giu
GAA
Lys
AAG
Arg
AGA
lie
ATC
Arg
CGC
Lys
AAG
Giy
GGA
Her
TCT
Vai
GTT
Leu
CTG
Giu
GAG
His
CTG
Lys
AAA
Glu
GAG
Giu
GAG
It@t
ATG
Gin
CAG
Asn
AAC
S.r
TCT
Pro
CCC
Giy
GGT
Asp
GAC
Tyr
TAT
Phe
GGC
Asp
GAT
Lys
AAA
Giy
GGA
Ala
GCT
Ser
AGT
Trp
TGG
Lys
AAG
lie
ATC
Leu
CTG
5cr
AGC
Pbs
TTC
Giu
TCT
Ala
GCC
Leu
CTC
Val
GTG
Asp
GAT
Pro
CCA
Tyr
TAC
Asp
GAT
Leu
CTC
Val
GTC
L.u
TTG
Car
AGC
L.u
CGA
L.u
TTG
Pro
CCT
Lys
AAA
Glu
GAG
Giu
GAG
Asn
AAT
Giu
GAA
lie
ATC
tie
ATA
Asn
AAC
L.u
TTG
Pro
GCA
Val
GTT
Vai
GTC
Pbs
TTC
Met
ATG
Cys
TGC
Giu
GAG
Ii.
ATA
Ii.
ATT
Cys
TGT
Tb.r
ACC
Vai
GTA
Ii.
TTG
Leu
CTG
His
CAT
Leu
CTG
Giy
GGC
Lys
AAG
Vai
GTT
Asp
GAC
Ser
TCC
Asp
GAC
Ser
AGC
His
CAT
Leu
GGC
Tyr
TAT
Val
GTG
Trp
TGG
Lou
CTA
Pro
CCA
Giy
GGG
Gin
CAA
Tyr
TAT
Giu
GAG
Arg
CGG
Phe
TTT
Lye
CTG
Glu
GAG
Vai
GTT
Giu
GAG
Gly
GGA
Giu
GAA
Lys
AAA
Lye
AAG
Giu
GAG
Giy
GGA
Arg
CGG
Vai
GTT
Giy
AAA
Pro
CCT
Vai
GTT
Cys
TGT
Lys
AAG
lie
ATT
Trp
TGG
Leu
CTG
Thr
ACC
His
CAC
Vai
GTG
Asn
AAT
Arg
AGG
Pro
CCC
Asp
GAC
Vai
GTC
Thr
ACG
Asp
GAC
Leu
CTC
Giu
GAA
Pbs
TTC
Arg
AGG
Leu
CTC
8cr
TCC
Asp
GCT
Pro
CCG
Pro
CCT
Thr
ACC
Lou
CTG
Lys
AAG
Giy
GGA
Giy
GGA
Arg
CGC
Leu
CTC
lie
ATC
Giy
GGC
Ala
GGG
Leu
CTG
Ii.
ATT
Ser
AGT
Gin
CAG
Ala
GCA
Giy
GGG
Phe
TTC
Leu
CTT
Lye
AAG
5cr
TCC
tie
ATC
Aia
GTC
Ser
AGC
Lou
CTC
Arg
CGG
Cys
TGC
Vai
GTG
Arg
AGG
Met
ATG
His
CAT
Asn
AAC
Giy
GGA
Leu
CTA
Aia
CGC
Ala
GCT
Her
AGT
Arg
CGC
Ii.
ATC
Vai
GTG
lie
ATC
Asn
AAC
Vai
GTT
Car
TCT
Thr
ACT
Giy
GGG
6cr
CGG
His
CAT
Lys
AAG
Ii.
ATC
Thr
ACA
Vai
GTG
Gin
CAA
Gin
CAG
Giy
GGA
Giu
GAG
Pro
CCC
Thr
ACT
Giu
GCC CTC
Asp Gin
GAC CAG
Val Leu
GTT TTG
Pro Gly
CCT GGC
Lou Met
TTG ATG
Ser Pro
TCG CCT
Pro Leu
CCT CTG
XXX Giy
CGN GGA
Vai Leu
GTC CTC
Aan Gin
AAT CAG
Ii•Gin
ATC CAG
Ala His
GCC CAT
Ala Asp
CAT
Leu
CTG
Arg
CGG
Her
AGC
Trp
TGG
Ser
TCC
Ala
GCC
Ala
GCC
Gin
CAG
Thr
ACT
Mn
AAT
Giu
GAA
Arg
66
88
110
132
154
176
198
220
242
264
286
308
330
352
374
17 32 TTC AAG AAG CAT TTT GAA TTG CCA ATT TTG AAG GGT CGA GAC GCT GCT GCT AGT GAG GCA GAC AGG
Gin Lou
Giy Giu Giu Arg
Leu Arg Giu Leu Thr Her
Ii. Vai Asn Arg Cys Lou
Ii. Arg Arg Thr
396
1798 CAG CTA GGA GAG GAG CGG CTG CGG GAG CTC ACC AGC ATT GTG AAT AGA TGC CTG ATA CGG AGG ACT
1864
6cr Asp Ii. Leu 6cr Lye Tyr Lsu Pro Val Lye XiS Giu Gin Vai Vai Cye Cye Arg L.u Thr Pro
TCT GAT ATC CTT TCT AAA TAT CTG CCT GTG AAG ATT GAG CAG GTC GTT TGT TGT AGG CTG ACA CCC
Leu Gin Thr Giu Lou Tyr Lye Arg Ph. Leu Arg Gin Ala Lys Pro Ala Giu Glu L.u L.u Glu Giy
418
440
193 0 CTT CAG ACT GAG TTA TAC AAG AGG TTT CTG AGA CAA GCC AAA CCG GCA GAA GAA TTG CTT GAG GGC
Lye Met
8cr Vai
Ser 5cr Leu 6cr 5cr
Ii. Thr
6cr Leu Lye Lye Leu Cye ken
His Pro Ala Leu
462
1996 AAG ATG AGT GTG TCT TCC CTT TCT TCC ATC ACC TCG CTA AAG AAG CTT TGT AAT CAT CCA GCT CTA
2062
Ii. Tyr Asp Lye Cye Vai Giu Giu Giu Asp Giy Phe Vai Giy Ala Leu Asp Leu Phe Pro Pro Giy
ATC TAT GAT AAG TGT GTG GAA GAG GAG GAT GGC TTT GTG GGT GCC TTG GAC CTC TTC CCT CCT GGT
Tyr 5cr Ser Lye Aia Lou Glu Pro Gin Leu Ser Gly Lye Met Leu Vei Leu Asp Tyr Ii. Leu Ala
484
506
2128 TAC AGC TCT AAG GCC CTG GAG CCC CAG CTG TCA GGT AAG ATG CTG GTC CTG GAT TAT ATT CTG GCG
2194
2260
Vai
GTG
Ph.
TTT
Lye
Thr
ACC
Giu
GAG
Arg
Arg
CGA
Lye
AAG
Ala
8cr
AGC
Leu
CTG
Lye
Arg
CGT
Cys
TGC
Vai
5cr
AGC
Arg
CGT
Val
5cr
AGT
Ala
GCC
Giu
Asp
GAC
Arg
CGA
Arg
Lye
AAA
Arg
AGG
Phe
Vai
GTA
Tyr
TAC
Aen
Vai
GTG
Leu
TTA
Ser
Leu
CTG
Tyr
TAC
Pro
Val
GTG
Vai
GTC
8cr
Her
TCG
Arg
CGC
Ser
ken
AAT
Leu
CTG
Pro
Tyr
TAC
Asp
GAT
Asp
Thr
ACC
Giy
GGC
Phe
Gin
CAG
Thr
ACG
Vai
Thr
ACT
Met
ATG
Ph.
Leu
TTG
Ser
TCC
Met
Asp
GAT
lie
ATT
L.u
Leu
CTC
Lye
AAG
Ser
528
550
572
2326 AAG CGA GCC AAG GTT GTA GAA CGC TTC AAT AGT CCA TCG AGC CCT GAC TTT GTC TTC ATG CTG AGC
6cr
2 392 AGC
Trp
2 45 8 TGG
Tyr
2524 TAT
Lye
Lye
AAA
ken
AAC
I]..
ATC
Ala
Aia
GCT
Pro
CCA
Tyr
TAC
Leu
Giy
GGG
Ala
GCC
Arg
CGC
Her
Giy
GGC
Asn
AAT
Leu
CTG
Her
Cye
TGT
Asp
GAT
L.u
CTG
Cye
Giy
GGC
Giu
GA.A
Ser
TCT
Vai
Leu
CTC
Gin
CAA
Ala
GCA
Vai
Asn
AAT
Ala
GCC
Giy
GGG
Asp
Lou
CTC
Met
ATG
Thr
ACC
Giu
Xi.
ATT
Ala
GCC
Ii.
ATT
Glu
Giy
GGG
Arg
CGG
Glu
GAG
Gin
Ala
GCT
Vai
GTC
Giu
GAG
Asp
Asn
AAC
Trp
TGG
Lye
AAG
Vai
Arg
CGG
Arg
CGA
Ii.
ATC
Giu
Leu
CTG
Asp
GAT
Ph.
TTC
Arg
Val
GTC
Gly
GGT
Gin
CAG
His
Met
ATG
Gin
CAA
Arg
CGT
Ph.
Phe
TTT
Lye
AAG
Gin
CAG
Ser
Asp
GAC
Lye
AAG
Ser
AGC
Leu
Pro
CCT
Thr
ACT
Hie
CAC
Giy
Asp
GAC
Cye
Lye
AAG
Giu
594
616
638
660
2 59 0 AAG GCA CTG AGC AGC TGT GTG GTG GAT GAG GAG CAG GAT GTA GAG CGC CAC TTC TCT CTG GGC GAG
2656
Leu Lye Giu Leu Phe lie L.u Asp Giu Ala Ser Leu Her Asp Thr His Asp Arg L.u His Cye Arg
TTG AAG GAG CTG TTT ATC CTG GAT GAA GCT AGC CTC AGT GAC ACA CAT GAC AGG TTG CAC TGC CGA
Arg Cye Vai Aen Ser Arg Gin Xi. Arg Pro Pro Pro Asp Giy Ser Asp Cye Thr Her Asp Leu Ala
682
704
2722 CGT TGT GTC AAC AGC CGT CAG ATC CGG CCA CCC CCT GAT GGT TCT GAC TGC ACT TCA GAC CTG GCA
Giy
Trp Am
Hie Cye Thr Asp Lye Trp Giy Leu Arg Asp Giu Vai Leu Gin Ala Aie Trp Asp Ale
726
2788 GGG TGG AAC CAC TGC ACT GAT AAG TGG GGG CTC CGG GAT GAG GTA CTC CAG GCT GCC TGG GAT GCT
2854
Ala Her Thr Aia Ii. Thr Ph. Val Ph. His Gin Arg Her His Glu Giu Gin Arg Gly Leu Arg Stop
GCC TCC ACT GCT ATC ACC TTC GTC TTC CAC CAG CGT TCT CAT GAG GAG CAG CGG GGC CTC CGC TGA
747
29 20 TAACCAGCTGGTCTGGGTGTAGCTCTTAGAGGAAGGAGATAGGGAAAAGGGGCTCCTTGCTCCACAGGGCCCTGTTGAATTTTGTTC
30 07 TTTGGGAGAAAATCATCAAGAAGGGCTGCATGATGTTTGCCCAAAATTTATTTTATAAGAAAAACTTTTTTGGTTAA@AA@@G@
3094
@@GGTATGAAAGGGTTAAAAAAAAAAAAAA.AAAAAAAJ,AAAA
Fig. I . Nucleotide sequence and predicted amino acid sequence of hRAD54. The sequence is in the one letter code (boldface) below coding sequence. A potential polyadenylation
signal sequence is underlined.
identified. Both BAC clones were subsequently found to contain the
entire hRAD54 genomic locus. We compared the hRAD54 cDNA
with cloned genomic DNA to determine the genomic organization of
the hRAD54 locus (Fig. 2). The gene spans a minimum of 25 kb of
DNA and consists of at least 18 exons. Length and relative position of
FIRADS4 exons and introns are summarized in Table 1. Exon 1 is the
1 2
3
4
5
6 7
8
largest exon, spanning the first 677 bp of the pBS cDNA. We cannot
exclude the presence of additional untranslated exons upstream of
exon 1 because Northern analysis appears to suggest a larger minor
transcript of4.0 kb in testis (Fig. 3). RACE amplification ofthe 5'-end
suggests that the approximately 3.2-kb transcript starts at nucleotide 1
of our 3135 cDNA clone (data not shown). Within exon 1 lies the
9
10
11 12 13 14 15 16
1718
Fig. 2. Physical map of hRADS4. Exons are represented by boxes, introns by lines. Exon numbers are shown above each exon.
2380
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HUMAN HOMOLOGUE OF RADS4
Table 1 Length and position of exons and introns of the hRAD54 gene
DNA sequence analysis of this altered exon product revealed a C-to-T
transversion, which resulted in a missense alteration (R587W). Be
ExonLength
(bp)1―>6771—6771103287678—764213003120765—88431100461885—945411005136946—108154426701082—1151611572891152—1440724681251441—15658520091511566
(bp)Position
cDNAIntronLength
cause this alteration is heterozygous with the wild-type sequence, it is
likely to represent a sequence polymorphism, although we cannot rule
out a dominant-negative mutation. Interestingly, some tumors ap
peared to show LOH of one of the two alleles (data not shown),
confirming the involvement of this chromosomal region in breast
carcinomas.
on
Discussion
We have cloned the human homologue of the RAD54 gene and
mapped it to chromosome lp32. The hRAD54 gene encodes for a Mr
93,000 protein containing 747 amino acids and belongs to the SNF2
superfamily of DNA and RNA helicases, the members of which are
involved in various aspects of DNA replication, repair, and gene
expression. Proteins from this family share homology within seven
conserved helicase motifs and in regions outside these motifs (35).
The first motif(motifl) corresponds to the A-Box of the NTP-binding
motif and lies within exon 7 of hRAD54. The consensus for motifs Ia
and II, the latter corresponding to the B-Box of the NTP-binding
motif, lies in exons 7 and 8—9of hRAD54, respectively. Conserved
sequence for the remaining motifs are encoded by exons 9 (motif III),
exons 10—11 (motif IV), exon 16 (motif V), and exons 17—18(motif
VI) of hRAD54. A multitissue Northern blot revealed that hRADS4
was expressed as a 3.2-kb transcript primarily in testis and thymus,
with lower (but detectable levels in small intestines, colon mucosa,
breast, and prostate; Fig. 3). The function and/or translation of the low
level 4.0 mRNA transcript observed in testis is unknown. This cx
aThe5'endboundary
ofexon
1wasnotdetermined.
S
//
RAD54
Table 2 Sequences ofprimers ampl@fvingindividual hRAD54 exons
Primer sequences
1
5‘
-CTAATCTCTCGTCTCGGC -3'
5'-TGACCCAGGGCTATTCCCA- 3'
5 ‘
-TAGGCTGCAGGATCCTTG- 3'
5 , -CTAGAAACCAAATCCTGGC-3'
5,-CCTGGCACTTAATAAGCAC -3'
5,-TGACTGGGCACAGACATAC -3'
273
5, -CCATAACATCTCCAGTCAG
-3'
164
2
Fig. 3. Multitissue Northern blot analysis of hRAD54. A 3.2-kb transcript is detected
primarily in testis and thymus tissues with lower levels ofexpression
3
in small intestine and
colon mucosa. A minor 4.0-kb transcript can be identified in testis. PBL, peripheral blood
4
5,-CAGGCACACGTACATATG
lymphocytes.
presumptive ATG start codon at position 675—677,immediately be
fore the 5'-end boundary ofintron 1. Introns 2—4,8—10,and 16 are the
largest, and their sizes were determined by gel electrophoresis of
inter-exon PCR products. The size of the remaining introns was
determined by direct sequencing. All of the intronic splice sites
confirm the GT-AG rule. We have evaluated the 5'-untranslated
region (Gen.Bank accession number) for consensus transcription fac
tor binding sites. A consensus TATA-Box2 was found at position
— 1.01
kb
relative
to
the
cDNA
initiation
site.
In
addition,
1 1
Spi
5 , -CTGGAGCTCCTAAACATAG-
6
5 , -GCGTGCATATACAGAGAAC-3'
5‘
-TTGCCCATGTGTGAGCAC -3'
5'-ACTTTGGCACCTACGCTG-
7a
5'-AGCGTAGGTGCCAAAGTG5 ,-AGTTCTTCACCAGGCTGG
7b
8
9
257
3@
238
207
3'
3'
240
-3'
5‘
-CTGCAGTGCATCACATTGA- 3'
5 , -TAAGAAAGCAGCAGGCTG-3'
5'-AGATTCTGAATTGTTCCC-3'
5 , -GGCAATACTCAGTGAAGAG-3'
253
5, -TACCGTATAGGGAATGCC
-3'
253
S‘
-AAAGACAAGGCAGGGCTC
10
208
-3'
5
II
sites, 1 AP1 site, and 1 NFl consensus sites were identified in this
1.5-kb upstream region.
Expression of an approximately 3.2-kb hRAD54 transcript was
primarily detected in testis and thymus with low levels of expression
in small intestines and colon mucosa (Fig. 3). In addition, low level
expression was observed in breast and prostate (data not shown). To
evaluate whether the hRAD54 gene was altered in tumors showing
LOH at lp32, genomic DNAs from 17 breast tumor cell lines and 20
sporadic breast tumors were analyzed by SSCP. Table 2 summarizes
the primers used for the amplification of each single exon. Exons 7
and 18 where divided into two segments to generate PCR products
suitable for SSCP. A single heterozygous alteration in the pattern of
the bands was found in exon 16 of the BT-20 breast tumor cell line.
Length of
product (bp)
Exon
5‘
-CTGCCATCACTAGCTGTG5,-AAGGATTGGCCATGGATG-
231
-3'
3'
3'
5'-GCATGGCAATTTTACCAGC3'
259
I74
5' -TTCAGGAGCTAGGCTTTG- 3'
12
5'-ATCAAGGTGTTCTCAGAGG5 ,-TTGCTCACTCTCACAGAG
3'
-3'
241
13
5‘
-CTAGTGAACACTGAAGTGG -3'
5' -GAAACAGGACAGCCCTAG- 3'
5‘
-AAGAAGCCTGGGCCATTG- 3'
5 , -AGACAGACAGTAGGGGAG-3'
258
14
15
5‘
-TCCCCCTAATCATTGAAGC-3'
5 ,-CTGATGATACTGCATTGG
16
17
l8a
l8b
293
257
3'
5'-AGTGCCCTAACCATTATC- 3'
5' -TGGAAGACGAAGGTGATA- 3'
5'-CCACTGCACTGATAAGTG- 3'
5 ,-ATGCAGCCCTTCTTGATG-
176
-3'
5‘
-CAGGATCCCAGTTTAGGC -3'
5,-CTGAAGATCTCGTTCCATG -3'
5 ‘
-ATGGCTAAGCGCTGTATC -3'
5'-ACTGTGTGGGTAGCTTAG-
244
258
242
3'
2381
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HUMAN HOMOLOGUE OF RAD54
pression pattern is generally typical of genes involved in DNA re
combination and repair (45, 46).
DNA helicases unwind duplex DNA, forming single-stranded DNA
that is accessible for replication, recombination, and repair. They play
pivotal roles in ensuring fidelity in the transmission of genetic infor
mation. Indeed, mutations in genes with proven DNA helicase activity
have been implicated in a variety of genetic disorders, including
cancer-prone syndromes like xeroderma pigmentosum and Bloom
syndrome. The hypothesis that loss of function of genes that safeguard
genome stability accelerates the tumorigenic process through accu
mulation of genetic alterations is supported by the finding that loss of
MSH2 function in homozygous mice predisposes the animals to
develop tumors (47—49).
Several groups have identified deletions involving most or all of
the short arm of chromosome I are the most frequent genetic
alterations found in tumors of neural crest origin like pheochro
mocytoma and medullary thyroid carcinoma (30—31). Further
more, the loss of genetic material at lp32—pter is detected in
meningiomas (32, 34), and this loss appears correlated to tumor
progression. In breast cancer, four discrete regions of allelic im
balance have been defined, suggesting the presence of several
genes involved in tumor development. One of these regions is
located at chromosomal band lp32, where LOH was detected in
47% of the tumors analyzed. We tested whether hRAD54 was
altered in breast tumors because we found it to be located in one of
the most common regions of LOH in this tumor type. We found no
clearly identifiable alterations of hRAD54 coding or intron/exon
sequence. These results appear to exclude the involvement of
hRAD54 in the pathogenesis of sporadic breast cancer. However,
we cannot exclude the possibility that there are alterations in the
5'-untranslated region that result in altered expression of hRAD54.
The role of hRAD54 in other tumors with deletions at lp32
remains still to be defined.
In conclusion, we have cloned the human homologue of the RAD54
gene and mapped it to a region of importance in a variety of solid tumors.
The structural characterization of its genomic locus will help in under
standing its possible involvement in malignancies in the attempt to link
repair pathways with tumor development and progression.
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phage
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Characterization of the Human Homologue of RAD54: A Gene
Located on Chromosome 1p32 at a Region of High Loss of
Heterozygosity in Breast Tumors
Debora Rasio, Yoshiki Murakumo, David Robbins, et al.
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