ABSTRACT
The full length-mouse XPC cDNA contains a 2703 bp orf which encodes a polypeptide
containing 900 amino acids. Overall, there is 75% identity in nucleotide
sequence and 73% identity in amino acid sequence between mouse and human genes.
The C-terminal half is more conserved (80%) than the N-terminal half (65%). Northern analysis has revealed a constitutive
expression pattern for both human and mouse transcripts in various tissues
examined. However, high level expression was observed in liver, testis and
kidney in both species. The human XPC gene was cloned from a cosmid library and
the full-length gene was found to span ~24 kb. Analysis of the genomic structure indicated that the transcribed sequence
is divided into 15 exons.
One of the principal DNA repair pathways that has the capacity for eliminating a
wide range of structurally unrelated lesions is referred to as nucleotide
excision repair (NER; for reviews see
1
-
4
). Defects in NER have been shown to give rise to a number of human genetic
syndromes which include xeroderma pigmentosum (XP), Cockayne's syndrome (CS)
and trichothiodystrohpy (TTD;
1
). Eleven genetic complementation groups have been identified among these three
syndromes and eight of these genes have now been cloned (
3
,
5
).
The XPC gene is believed to be involved in an early stage of the incision
process. It is known that XPC forms a stable complex with the yeast RAD23
homologue HHR23B (
6
) and interacts with the transcription/repair factor TFIIH (
7
,
8
). The XPC gene binds non-specifically to single-stranded DNA with high affinity. Therefore, a plausible role for XPC
is to stabilize the bubble structure, presumably created by the helicase
activity of TFIIH, around the lesion. Unlike most other XP cells, XPC cells are
deficient only in the overall genome repair while the preferential repair of
the template strand of actively transcribed genes is normal (
9
). This suggests that XPC can be replaced by another factor(s) or is not
required at the site of a stalled-RNA pol II complex. In a fully reconstituted
in vitro
NER system, Mu and colleagues were able to show that excision of lesions can
occur with a certain substrate without XPC even in the absence of transcription
(
10
). In order to elucidate the precise function of XPC it is important to analyze
its peptide structure and to identify functional domains through sequence
analysis of its homologues. Previously, we cloned the human XPC cDNA by
functional complementation and found that XPC had significant homology to the
Saccharomyces cerevisiae
Rad4 protein, particularly in the C-terminal end (
11
). Cloning of the
Drosophila
homologue of human XPC confirmed the homology to RAD4 (
12
). Additional homologues of XPC are required to define conserved and possibly
functional domains. Analysis of the XPC gene structure will facilitate studies
on the characterization of XPC mutations and on the control of its
transcription. Here, we describe the cloning and characterization of the mouse
XPC cDNA, the tissue- specific expression of the human and mouse transcripts, and the genomic
structure of the human XPC gene.
An 8 1/2 day C57BL mouse embryo cDNA library in vector [lambda]gt10 was screened with a human XPC probe. Pre-blotted nylon membranes with mouse and human polyA
+
RNA from multiple tissues were purchased from Clontech and each hybridized with
random primed mouse and human XPC cDNA probes.
Sequence analysis was performed with the Bestfit program from the Genetics
Computing Group (Genetics Computer Group, Madison, WI, USA). Other sequence
analysis was performed with the Lasergene Navigator program (DNASTAR Inc.).
A CEPH YAC library was screened by PCR (
13
). A cosmid library of the YAC DNA was constructed in the sCOS-1 vector (
14
).
Five clones from the screening of the cDNA library formed an overlapping contig
and the merged DNA sequence of the mouse XPC covered residues 20-3220 relative to the human sequence. The mouse XPC gene encodes a
transcript of ~3.8 kb based on Northern blot analysis (see below). We obtained 3030 bp of
the cDNA sequence and the longest orf consists of 2703 bp, corresponding to a
predicted polypeptide of 900 residues (GenBank accession no. U27398).
Human and mouse XPC share high levels of homology at both the nucleotide and amino acid sequence levels. Overall, the nucleotide sequences exhibit an 81% homology and the peptide sequences show a 74% identity
and a 85% similarity. However, the C-terminal half of the XPC protein is more highly conserved than the N-terminal half (80.3% versus 66.3% identity). The extreme N-terminus is particularly unconserved which may explain why
this region is dispensable in the human protein (
6
). The deduced mouse XPC polypeptide has a calculated molecular mass of 101 kDa.
Expression of XPC transcripts in various tissues were examined by Northern blot
analysis (Fig.
1
). Mouse XPC mRNA migrates at ~3.8 kb. Although both human and mouse XPC were expressed in all organs and
tissue types examined, the level of transcription varied to a large extent. In
both cases, when the signals were normalized by densitometry against an
internal [beta]-actin control (Fig.
4
c and d), liver, testes and kidney were found to have much higher levels
compared with cardiac, skeletal muscle, lung or brain.
Table 1
.
Organization of mouse XPC exons
Screening by PCR of a CEPH genomic YAC library yielded nine clones. Five were
found to contain the XPC 5' UTR as determined by Southern blotting. Further analysis by florescence
in situ
hybridization analysis (FISH) revealed two clones containing contiguous inserts.
The length of the XPC gene (24 kb) and the restriction enzyme map were
determined by Southern analysis (Fig.
2
). Sequencing of DNA was performed to cover the entire coding sequence to
elucidate the XPC genomic structure. The human XPC gene was found to be divided
into a total of 15 exons (Table
1
). Of the 14 donor/acceptor splice sites, 11 are consistent with the consensus
AG/GT sequence, however, three are comprised of the sequences AG/GG, GA/AT and
AT/GT.
Recent analysis of excision repair at the level of the gene has indicated that
XP-C cells are deficient in global repair, but not transcription coupled
repair (
9
). In normal rodent cells, lesions in the template strand of actively
transcribed genes are repaired efficiently, however, the non-transcribed portion of the genome is inefficiently repaired compared with
human cells. These findings have led to the suggestion that XPC may not play an
important role in NER in rodent cells. However, recent results have shown that
an XPC knockout mouse is highly sensitive to the effects of UV irradiation (
15
). In addition, the data we present here show a high degree of homology (85%
similarity) at the amino acid level between mouse and human XPC, suggesting
that they are likely to have similar biochemical functions. Among all
homologues of XPC sequenced to date, which include
S.cerevisiae
(RAD4;
16
),
Drosophila
(
12
), mouse and human, the most highly conserved region lies between amino acid
residues 517 and 867 in the human protein. Cloning of the mouse homologue will
aid further efforts to identify highly conserved and functional domains in XPC.
We thank David Cheo for sharing his unpublished results and Craig Chinault for his assistance in isolating XPC YAC clones. The DNA
sequencing was performed in the MDACC core facility supported by grant CA16672
from the US National Cancer Institute (NCI). This work was supported by grant
CA52461 from NCI.
Exon
Position
Size (bp)
1
-105-102
208
2
103-299
197
3
300-412
113
4
413-520
108
5
521-779
259
6
780-900
121
7
901-990
90
8
991-1871
881
9
1872-2033
162
10
2034-2115
82
11
2116-2250
135
12
2251-2420
170
13
2421-2514
94
14
2515-2604
90
15
2605-3453
849
REFERENCES
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