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© 1992 Oxford University Press
Nucleic Acids Research, Vol. 20, No. 17
4417-4421
Structure of the human DNA repair gene HAP1 and its
localisation to chromosome 14q 11.2-12
Craig N.Robson, Daniel Hochhauser, Randa Craig, Katrina Rack1, Veronica J.Buckle1
and Ian D.Hickson*
Imperial Cancer Research Fund and 1 MRC Molecular Haematology Unit, Institute of Molecular
Medicine, University of Oxford, John Radcliffe Hospital, Oxford 0X3 9DU, UK
Received June 26, 1992; Revised and Accepted August 6, 1992
EMBL accession no. X66133
ABSTRACT
Apurinic/apyrimkJinlc (AP) sites are pre-mutagenlc DNA
lesions which occur spontaneously and following
exposure of cells to ionising radiation or chemical
mutagens. HAP1 (Human AP endonuclease 1), the
major enzyme in human cells initiating repair of AP
sites, shows strong sequence homology to DNA repair
enzymes from bacteria, Drosophila and other
mammalian species. We have cloned the HAP1 gene
and determined its complete nucleotide sequence. The
site of transcription initiation has been mapped to 452
bp upstream of the ATG initiation codon in the genomic
DNA. The HAP1 gene consists of five exons and is
unusually small (less than 2.6 kb from transcription
initiation site to polyadenylation sequence) with 54%
of the protein coding region and the entire 3'
untranslated region contained within a single exon. The
first exon is non-coding. Regions of three exons show
sequence homology to the E.coli xth (exonuclease III)
gene. Using In situ hybridisation, the HAP1 gene has
been localised to human chromosome 14q 11.2-12.
shows strong sequence similarity to DNA repair enzymes not
only from other mammalian species (11,12), but also from
Drosophila (13) and bacteria (14,15). The predicted bovine
(BAP1) and human (HAP1) protein sequences show 93% amino
acid identity (9,11), while HAP1 shows 27% identity with E. coli
exonuclease IE protein (9,14), the best characterised enzyme in
this homologous group.
E. coli xth mutants are deficient in exonuclease HI and show
hypersensitivity to a variety of DNA damaging agents, despite
the presence of a second AP endonuclease, called endonuclease
IV, which can partially compensate for a lack of exonuclease
HI (6,7). Evidence for functional overlap between the HAP1
protein and bacterial AP endonucleases came from the finding
that expression of the HAP1 cDNA could overcome DNA repair
and mutagenesis defects in xth mutants and in xth nfo
(endonuclease IV) double mutants (9).
To gain insight into both the molecular mechanisms of HAP1
gene regulation and the possible role in human pathology of
defects in the HAP1 protein, we have isolated and characterised
the HAP1 gene and localised its site in the human genome to
chromosome 14q.
INTRODUCTION
Apurinic/apyrimidinic (AP) sites in DNA arise spontaneously at
physiological pH and following exposure to certain DNA
damaging agents (1-5). These sites of base loss are noninstructional for DNA polymerases and are consequently premutagenic lesions which may give rise to base substitutions. AP
sites are recognised by DNA repair enzymes known as AP
endonucleases, the major class of which (class II) cleave the
phosphodiester backbone 5' to the AP site via a hydrolytic
mechanism. In contrast, class I enzymes act as /3-elimination
catalysts and cleave 3' to the AP site (for reviews see 6,7).
Following incision by a class II AP endonuclease, the baseless
sugar/phosphate group is excised (8), the resulting gap filled by
a DNA polymerase and the nick sealed by a DNA ligase, thus
restoring the correct DNA sequence.
The major AP endonuclease in human cells, designated HAP1
(human AP endonuclease i ) , is a 35.5 kDa protein (9,10) which
* To whom correspondence should be addressed
MATERIALS AND METHODS
Isolation of the HAP1 genomic gene
A human genomic DNA library (kindly provided by Dr.
J.Trowsdale, ICRF, London) was screened using a fragment of
the HAP1 cDNA comprising the protein coding region.
Approximately 100,000 bacteria representing the library were
plated at 50,000 colonies/20 cm2 Bio-assay dish (Nunc) and
colonies transferred to HyBond-N membrane (Amersham). The
HAP1 probe was labelled by random priming using
[a-32P]-dCTP and Klenow polymerase. Hybridisation and filter
washing were performed according to manufacturer's instructions
with a final wash of 0.1 xSSC/0.1 % SDS at 65°C for 10 minutes.
Secondary and tertiary rounds of screening were conducted
similarly, with densities of 50-500 colonies per 82 mm filter.
Cosmid DNA, isolated from four positive clones, was cleaved
with restriction enzymes, transferred to HyBond-N and probed
4418 Nucleic Acids Research, Vol. 20, No. 17
with the HAP] cDNA. A 7 kb Hindm fragment and two PstI
fragments of 1.9 and 2.3 kb were subcloned into pUC18 and
used as templates for DNA sequencing.
INTRON
(Splice
acceptor)
INTRON
(Splice donor)
E l :ON
-I (0.202)
AGTAGG
(0.182)
(0.210)
Nucleotide sequencing
Sequencing was carried out by the dideoxy chain termination
method using Sequenase (USB Corporation). Multiple synthetic
oligonucleotide primers were used to sequence both strands of
the HAP I gene completely.
gtctgtaa GCAACG
I (0.126)
GGACAG
ttttatafi AGCCAG
II (0.188)
TTAGAT fltgagtgg (0.565)
acttacag TGGGTA
III (0.193)
GCATAG gtgagacc (0.130)
RNA isolation
Total cellular RNA was isolated from the human cervical
carcinoma cell line HeLa essentially by the method of
Chomczynski and Sacchi (16).
ttctatag GCGATG
IV (0.776)
Ribonuclease protection analysis
The probe was prepared by cloning a 529 bp HindlH-Pstl
fragment located 5' to the ATG initiation codon into pBluescript
(Stratagene) and digesting at the Xbal site in the polylinker.
Following addition of 0.5 ng Xbal-digested template to 10 mM
DTT, 0.4 mM NTP's, 5 /tl [a- 32 P]-CTP, 40u RNasin and 40 u
T3 RNA polymerase (Boehringer Mannheim), in a total volume
of 20 fd, the mixture was incubated at 37°C for 30 minutes. 35 u
DNase I were added and the sample incubated for a further 15
minutes at 37 °C. The sample was then spun through a G50
Sephadex column to remove unincorporated nucleotides.
Approximately 0.5 X106 cpm were added to 20 jtg total RNA
and the mixture incubated for 16 hours at 55 °C. Following
hybridisation, RNA-RNA hybrids were digested for 30 mins at
30°C in a solution containing 40 /tg/ml ribonuclease A and
2/ig/ml ribonuclease Tl. Reactions were stopped by addition of
SDS to 0.5% and proteinase K to 125/tg/ml. The sample was
vortexed in an equal volume of phenol/chloroform (50:50 mix),
ethanol precipitated, resuspended in gel loading buffer (80%
formamide, lmM EDTA, pH 8.0, 0.1% xylene cyanol, 0.1%
bromophenol blue) and run on a 6% polyacrylamide DNA
sequencing gel. The gel was then dried down and exposed to
x-ray film.
Primer extension analysis
A 36 mer oligonucleotide was synthesised corresponding to
positions 70 through 105 of the HAP] cDNA sequence (9, 17)
in the antisense orientation.
5' CTT AAT TAA GGG TCC TGA CTC AAG CTT GCC.GTT CAG 3'
Total cellular RNA (20/tg) was co-precipitated in ethanol with
105 cpm of oligonucleotide end-labelled with [y^PJ-dATP using
8u T4 polynucleotide kinase (Boehringer Mannheim). Samples
were resuspended in hybridisation buffer (80% deionised
formamide, 40 mM PIPES, pH 6.4, 400 mM NaCl, lmM
EDTA) heated to 90°C for 5 minutes and hybridized at 30°C
for 2 hours. Samples were then ethanol precipitated and
resuspended in a solution containing 50mM Tris-HCl, pH 8.3,
75mM KC1, 30mM MgCl2 and 200 units of murine moloney
virus (MMV) reverse transcriptase, in a reaction volume of 30/tl.
Extension reactions were for 1 hour at 37 °C before termination
by heating to 100°C for 5 mins in the presence of 0.1M NaOH.
Following the addition of 3/il 1M Tris-HCl, pH 7.5, the DNA
was ethanol precipitated and resuspended in 20/tl denaturing dye
buffer (lOmM EDTA, pH 8.0, 0.1% xylene cyanol, 0.1%
bromophenol blue in formamide). Products were run on a 6%
polyacrylamide/urea gel, and the gel dried and exposed to x-ray
poly A tall
Figure 1. Intron/exon structure of the HAP I gene. The nucleotide sequence of
each intron/exon junction is shown separated by vertical lines. Intron sequences
are shown in lower case and exons in upper case letters. The consensus splice
acceptor and splice donor sequences are underlined. The sizes of the exons and
introns are shown in parenthesis.
film. Labelled products were sized by reference both to endlabelled Haem-digested <£X174 DNA and to a standard DNA
sequencing reaction.
In situ hybridization
Fluorescence in situ hybridization to normal male metaphases
was performed using biotin-labelled HAP] probes, as described
by Hirst et al. (18). Probes consisted either of the entire HAP I
cosmid or the 7kb Hindm fragment of HAP1. The hybridisation
mixes contained either 80ng of cosmid DNA or 300 ng of the
7kb fragment, together with 2.5 /ig COT1 DNA. After probe
detection with successive layers of fluorescein-conjugated Avidin
(Vector Labs.) and biotinylated anti-Avidin (Vector Labs.), the
slides were mounted in antifade (Vector Labs.) containing 0.5
/tg/ml DAPI and 0.5 /tg/ml propidium iodide to obtain G bands.
A confocal laser microscope (Biorad MRC 600) was used for
the analysis. Images were collected in dual mode channel and
then merged.
RESULTS
Isolation of the HAP] gene
A human genomic DNA library was screened using a fragment
of the HAP] cDNA as probe. Cosmid DNA from positive clones
was digested with a variety of restriction enzymes and fragments
containing portions of the HAP] gene identified by Southern
blotting using the cDNA as probe. This analysis indicated that
most of the HAP] gene lay on a 7 kb Hindin fragment. This
fragment excluded only the DNA upstream of the Hindlll site
at positions 79-84 of the published cDNA sequence (9,17). This
upstream region was mapped to a 2.3kb Pst I fragment (data not
shown).
DNA sequence of the HAP1 gene
The nucleotide sequences of the entire 2.3kb Pst I fragment and
a portion of the 7 kb Hindin fragment were determined by the
dideoxy chain termination method. By reference to the previously
published cDNA sequence for HAP] (9,17), it was possible to
define the complete HAP] gene intron/exon structure. The gene
consists of five exons and four introns, with a total length of 2572
bp from the site of transcription initiation (see below) to the start
of the polyadenylation sequence. The complete sequence of all
exons and introns has been deposited with the EMBL library
Nucleic Acids Research, Vol. 20, No. 17 4419
EXON-I
EXONI
EXONII
EXONm
EXON IV
TGA
ATG
-TWWWN-
Flgure 2. Structure of the HAPl gene. The structure is shown drawn to scale. Exons are shown as boxes and are labelled above. Intervening introns are shown
as horizontal lines. The ATG initiation and TGA terminination codons are indicated. The hatched areas represent regions of homology with the E.cotixth (exonuclease
III) gene.
under the accession number X66133. The sequence of the exons
matches our published cDNA sequence (9,17) except for the
following; insertion of an extra G residue after C-187 in the 5'
non-coding region, a G to C change at position 887 (Pro223
remains unchanged), and a GC to CG switch at positions 927/928,
changing Ala237 to Arg. All of these base and amino acid
numbers refer to the previously published cDNA sequence (9,17).
Gene structure and sequences of intron/exon junctions
Figure 1 shows the sequences around the intron/exon junctions,
together with the sizes of exons and introns. Sequences at the
junctions match the consensus donor and acceptor splicing signals
(reviewed by 19,20). A structural map of the HAPl gene is shown
in Figure 2. The first intron is contained within the 5' non-coding
region of the HAPl gene, and thus the first exon (designated exon
—I) is non-coding and the ATG initiation codon is within the
second exon (designated exon I). The small overall size of the
gene results from the unusually short length of the introns, with
three out of four being < 220 bp. In contrast, exon IV is 776
bp long and contains 54% of the HAPl protein coding region
and the entire 3' non-coding region (9,17). The domain of the
HAPl gene which shows homology to the family of AP
endonucleases, including E.coli exonuclease m (9,14), is not
contained within a single exon, but forms part of exons II, HI
and IV (Figure 1).
Mapping of the transcription initiation site
The site of transcription initiation of the HAPl gene was mapped
by both primer extension and RNase protection analyses.
For primer extension analysis, a 36 mer oligonucleotide primer
complementary to the 5' end of the published cDNA sequence
(9,17) from positions 70 through 105 produced a poorly resolved
cluster of extension products centred around 165 nucleotides in
length, and a weaker doublet of 188/189 nucleotides (Figure 3).
With short exposure times of the autoradiogram, the cluster could
be resolved into at least 3 distinct bands. The product of 165
nucleotides corresponds to a start site in the genomic DNA 452
bp upstream of the ATG initiation codon (this includes the 182
bp intron in the 5' non-coding region). The size of the 5' noncoding region in the HAPl mRNA would therefore be 270
nucleotides.
To verify the primer extension data and to exclude the
possibility of other introns being present in the 5' non-coding
region, RNase protection analysis was carried out. A radiolabelled
probe extending from the HindTJI site at positions 79-84 in the
HAPl cDNA (9,17) to a point 529 nucleotides upstream was
used. This probe was hybridized to HeLa cell RNA, and the
mixture digested with RNases before running on a polyacrylamide
gel. The major protected fragment was calculated to be 116 bp
in length, but an additional larger fragment of 152 bp was
consistently observed (Figure 4). The bands migrating faster than
1 2 3 4 5 6 7
-
234
194
- 1 18
Figure 3. Determination of the HAPl gene transcription start site by primer
extension analysis. A radiolabelled oligonucleotide corresponding to positions 70
through 105 of the HAPl coding region was used as a primer in a reverse
transcription reaction using HeLa cell total RNA. The products were resolved
on a 6% polyacrylamide gel. Lanes 1-4, DNA dideoxy sequencing reaction;
Lane 5, primer extension products with HeLa cell RNA; Lane 6, primer extension
products with tRNA; Lane 7, 0x174 digested with HaelD. (molecular weight
markers). The major extension products are indicated by a large arrow and the
minor products by a small arrow.
the 116 bp fragment were not always observed and probably
represent degradation products of the major fragment. The sizes
of the protected fragments correspond to transcription start sites
424 bp and 460 bp upstream of the ATG initiation codon,
respectively, in the genomic DNA sequence. These distances take
into account the 182 bp intron present in the 5' non-coding region.
We arbitrarily assigned the transcription start site (position +1)
to the position defined by the major primer extension product
(452 bp upstream of the ATG in the genomic DNA sequence),
which also corresponds approximately to a product defined by
RNase protection analysis.
Structure of the 3' non-coding region of the HAPl gene
The nucleotide sequence of the 3' non-coding region of the HAPl
gene matches exactly the published cDNA sequence (9,17). The
only polyadenylation signal sequence within 240bp of the TGA
4420 Nucleic Acids Research, Vol. 20, No. 17
1 2
-234
- 1 94
-118
-
72
Figure 4. Determination of the HAP] gene transcription start she by ribonuclease
protection analysis. A 529 bp fragment in pBluescnpt was transcribed in the
antisense orientation. The RNA probe generated was hybridised with HeLa cell
RNA or with tRNA as control and the RNA:RNA hybrids digested with RNases.
The products were separated on a 6% polyacrylamide gel alongside size standards
(indicated on the right). Lane 1, RNA probe annealed to HeLa total RNA and
digested; Lane 2, RNA probe annealed to tRNA and digested. The major protected
fragment is indicated by a large arrow. The upper band, indicated by a small
arrow, corresponds to the start site identified by primer extension analysis.
translation stop codon is that previously identified in the cDNA
sequence, indicating a sequence of AAUAAAGAGCCAUAGUUUC(A)n for the 3' end of the HAP1 mRNA.
Chromosomal localisation of the HAP1 gene
Southern blotting using DNA from human:rodent hybrids
suggested that the HAP] gene lay on chromosome 14 (data not
shown). This was confirmed using the polymerase chain reaction
to amplify HAP 1 gene-specific DNA from a variety of hybrid
lines, including one containing chromosomes 14 and 18 as the
only DNA of human origin. To accurately map the gene location,
in situ hybridisation using a biotin-labelled HAP 1 probe was
performed. The result (Figure 5) shows specific hybridisation
to both chromatids of chromosome 14 localised to the long arm
at bands 11.2—12. Both the entire HAP1 -containing cosmid and
the 7kb Hindin subclone gave a specific signal at an identical
location on chromosome 14. A control probe (locus D14S24),
previously localised to chromosome 14, was used to confirm the
chromosomal assignment (data not shown).
DISCUSSION
We have isolated and completely sequenced the DNA repair gene
HAP1, which encodes the major AP endonuclease expressed in
human cells. The HAP1 gene is one of the smallest identified
Figure 5. In situ hybridization of human metaphase chromosomes using a HAP]
genomic DNA as probe. Specific hybridization to both chromatids of each
chromosome 14 at q l l . 2 - 1 2 is arrowed.
in the human genome with a size of —2.6 kb from the site of
transcription initiation to the site of polyadenylation. This is
principally because the gene contains only four introns of which
three are less than 220 bp in length. One of these introns lies
within the 5' non-coding region and thus the ATG initiation codon
is located within the second exon. Amino terminal amino acid
sequencing indicated that the proposed ATG initiation codon of
the HAP I gene has been correctly assigned (unpublished results).
It is common for the exons of genes in human cells to be
relatively short (around 250 bp or smaller). This may be to limit
the scope for gene rearrangements resulting from recombination
events, thus maintaining gene stability in coding regions. The
organisation of the HAP] gene is somewhat atypical in that while
three of the four coding exons are below 200 bp in length, the
fourth is nearly 800 bp long. This unusually long exon includes
54% of the HAP1 protein coding region and the entire 3' noncoding region. This gene structure cannot readily be explained
by the conservation during evolution of a particular domain of
the protein, as the region of homology between the HAP1 gene
and the E.coli xth gene (exonuclease III) (14) is interrupted by
two introns.
We have mapped the site of transcription initiation (cap site)
to a point 452 bp upstream of the ATG initiation codon. Of this
DNA, 182 bp is represented by the first intron, and thus the 5'
non-coding region of the HAP1 mRNA is calculated to be 270
nucleotides in length. This indicates that the published cDNA
(9,17) was truncated at the 5' end by 60bp. We have sequenced
the region 5' to the cap site where many of the sequences for
HAP] gene regulation would be expected to be located (21).
There is no sequence closely matching the consensus for a TATA
box at the appropriate distance from the cap site (for a review
of transcription factor binding sites, see 22). However, there is
a CCAAT box located 55 bp 5' to the cap site, although we have
no evidence that this is functional. Further work is required to
Nucleic Acids Research, Vol. 20, No. 17 4421
define the elements essential for regulation of HAP] gene
expression.
We have localized the HAP1 gene to chromosome 14q
11.2-12. In the absence of known human cell mutants defective
in HAP1 protein function, it is not possible to make definitive
comments on the phenotype of a HAP1 mutant. However, by
comparison with the phenotype of E.coli xth mutants (23, 24)
it seems likely that human cells defective in HAP1 enzyme
activity would show multiple abnormalities. A failure to
efficiently repair oxidative and chemical DNA damages would
be expected to lead to enhanced sensitivity to DNA damaging
agents and/or hyper-mutability. One could speculate that in
individuals this may lead to an accumulation of DNA damage
in tissues exposed to oxygen-derived free radicals, or to an
increase in susceptibility to cancer. To date, there are no genetic
disease gene loci conferring a phenotype consistent with such
abnormalities which have been mapped to the appropriate region
of chromosome 14q (25).
In summary, we have isolated and completely sequenced the
human DNA repair gene, HAP1, involved in cellular protection
against the lethal and mutagenic effects of reactive oxygen
species. HAP1 has an atypical intron/exon structure and is one
of the smallest genes identified in the human genome. The gene
maps to chromosome 14q 11.2—12. We are now in a position
to isolate human cell mutants deficient in HAP1 enzyme activity
and to identify the sequences controlling expression of this key
DNA repair gene.
ACKNOWLEDGEMENTS
We thank members of the ICRF Molecular Oncology Labortory
for useful discussions and Elizabeth Clemson for typing the
manuscript. This work was supported by the Imperial Cancer
Research Fund and the Medical Research Council.
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