hRAD30 Mutations in the Variant Form of Xeroderma Pigmentosum

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Science  09 Jul 1999:
Vol. 285, Issue 5425, pp. 263-265
DOI: 10.1126/science.285.5425.263


Xeroderma pigmentosum (XP) is an autosomal recessive disease characterized by a high incidence of skin cancers. YeastRAD30encodes a DNA polymerase involved in the error-free bypass of ultraviolet (UV) damage. Here it is shown that XP variant (XP-V) cell lines harbor nonsense or frameshift mutations in hRAD30, the human counterpart of yeastRAD30. Of the eight mutations identified, seven would result in a severely truncated hRad30 protein. These results indicate that defects in hRAD30 cause XP-V, and they suggest that error-free replication of UV lesions by hRad30 plays an important role in minimizing the incidence of sunlight-induced skin cancers.

Xeroderma pigmentosum (XP) patients are hypersensitive to sunlight, and they suffer from a high incidence of skin cancers. Cells from seven different XP complementation groups (A to G) are defective in nucleotide excision repair (1), whereas cells from the variant form of XP (XP-V) excise UV photoproducts at a normal rate (2). XP-V cells, however, are much slower than normal cells in replicating DNA containing UV photoproducts (3), and XP-V cell-free extracts are deficient in bypass replication of a cis-syn thymine-thymine (T-T) dimer (4). XP-V cells are hypermutable with UV light and exhibit an unusual mutational spectrum (5).The RAD30 gene of Saccharomyces cerevisiaefunctions in error-free bypass of UV lesions (6,7). RAD30 encodes a DNA polymerase, Polη, which can efficiently replicate past a cis-syn T-T dimer in template DNA, and Rad30 inserts two adenines across from the dimer (8). Here, we determine if the human homolog of yeastRAD30 is responsible for XP-V.

A human cDNA clone, H96386 [345 base pairs (bp)], that encodes a peptide with homology to the NH2-terminus of yeast Rad30 protein was used to screen a spleen cDNA library (9), and a single clone that contains a 3-kb cDNA insert was isolated. Sequence analysis (10) of this cDNA (11) (GenBank accession number AF158185) indicated that the protein encoded by the human gene displays significant homology to theS. cerevisiaeRad30 protein (Fig. 1A). We have named the human gene hRAD30. Excluding the intervening region from amino acid residues 452 to 606 in hRad30, the human and yeast Rad30 proteins share 23% identical and 53% conserved residues, and the two proteins have several highly conserved motifs throughout their length (Fig. 1A).

Figure 1

(A) Alignment of human Rad30 (hRad30) andS. cerevisiae Rad30 (yRad30) protein sequences. Identical and highly conserved residues are highlighted. Numbers in parentheses indicate amino acid positions; asterisks indicate stop codons. (B) Schematic representation of homology between human Rad30, S. cerevisiae Rad30, and S. cerevisiaeRev1 (yRev1). Unshaded boxes represent nonhomologous sequences and shaded boxes represent homologous sequences. Positions of highly conserved motifs are indicated. Gaps have been introduced for optimal alignment. Protein lengths are indicated by numbers on the right. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

The yeast REV1 gene encodes a deoxycytidyl transferase (12) that is homologous with Rad30 (6,13) (Fig. 1B). However, yeast Rev1 shares only 13% identical and 32% conserved residues with hRad30, and many of the conserved motifs in the COOH-terminal portion of yeast and human Rad30 are absent from Rev1 (Fig. 1B). For example, the Rad30 motif EH(M/A)DYH(F/L)AL is absent from Rev1 (Fig. 1B). In addition, the NH2- and COOH-terminal sequences of Rev1 are not found in yeast or human Rad30 (Fig. 1B). Thus, hRad30 is the human counterpart of yeast Rad30.

To determine whether defects in hRAD30 are responsible for XP-V, we sequenced (10) hRAD30 cDNA from eight different human XP-V cell lines and from five other control cell lines (14). RNA isolated from these cell lines was used to generate hRAD30cDNA, which was then amplified by polymerase chain reaction (PCR) (10). In the five control cell lines, which include a normal lymphoblast cell line, a HeLa cell line, and three cell lines derived from XPA, XPB, and XPC patients, we found no sequence changes in hRAD30. By contrast, eight distinct mutations were identified (Table 1) in the eight XP-V cell lines. The XP1CH and XP2CH cell lines are derived from two XP-V–affected sibs who are offspring of a first cousin marriage. Both have a C to T transition at nucleotide (nt) 376 (Fig. 2A) which creates a TAA nonsense codon that would result in a truncated protein of 125 amino acids. The XP115LO cell line, also derived from an individual who is an offspring of a first cousin marriage, carries a C to T transition at nt 1117 (Fig. 2B) that creates a TAA stop codon at position 372. The XP30RO cell line has a deletion of 13 bp (Fig. 2C) that would truncate hRad30 at position 35. The XP5MA cell line has a deletion of 104 bp (Fig. 2D) that would truncate hRad30 at position 220. In each of these five XP-V cell lines, only the respective mutant allele was observed. The XP6DU cell line has two different mutant alleles of hRAD30. One of these would result in the termination of hRad30 at position 69, and the other would result in the deletion of the Leu-75 residue from the protein. In cell line XPPHBE, one allele has a frameshift mutation that would terminate hRad30 at position 359. We have been unable to identify a mutation within the second hRAD30 allele in XPPHBE; however, this allele is expressed at about 20% the level of the mutant form (15). In cell line XP1SF, one hRAD30 allele has a C to T transition mutation that generates a TAG stop codon, resulting in a truncated protein of only 22 amino acids. In this cell line also, we have been unable to identify a mutation within the secondhRAD30 allele, but here too the wild-type allele is expressed at a lower level (∼10%) than the mutant allele (15).

Figure 2

Detection of hRAD30 mutations in XP-V cell lines. Numbers indicate nucleotide position in relation to +1 of the ATG initiation codon. The positions of mutational alterations are indicated. (A) The C→T mutation at nt +376 in XP1CH cell line. (B) The C→T mutation at nt +1117 in XP115LO cell line. (C) The 13-bp deletion from nt +104 to +116 in XP30RO cell line. (D) The 104-bp deletion from nt +661 to +764 in XP5MA cell line. The asterisk indicates the missing 92 nt of the normal hRAD30 sequence. The orientation of thehRAD30 sequence in (A) and (D) is different from that in (B) and (C).

Table 1

Mutations identified in XP-variant cell lines.

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In six of the cell lines (XP1CH, XP2CH, XP115LO, XP30RO, XP5MA, and XP6DU), we observed only the mutant form or forms of thehRAD30allele. Of the six different mutations present in these cell lines, five would result in premature termination of translation. The longest of these truncated proteins would contain only 372 amino acids and thus would retain only about 50% of the protein. Because the human and yeast Rad30 proteins share regions of homology beyond residue 372 of hRad30 (Fig. 1A), this hRad30 truncation is likely to result in the loss of function. The other four truncated proteins would contain only 35 to 220 amino acids of hRad30, and it is likely that they inactivate hRad30 function. Yeast Rad30 deleted beyond residue 340 has no DNA polymerase activity, and this mutation does not complement the UV sensitivity of the rad30Δstrain (16). One mutant allele of hRAD30 is deleted for the Leu-75 codon. This residue is identical in the yeast and human proteins, and it lies in a conserved region of the protein; thus, it may also affect hRad30 function. Two of the cells lines (XPPHBE and XP1SF) have a mutation in only one of the two chromosomes, and these mutations also would produce severely truncated proteins. However, in both these cell lines, the allele with no mutational alteration is expressed at a reduced level. Also, we have not excluded the possibility that these apparently wild-type alleles harbor a mutation in the 5′- or 3′-flanking regions that have not been sequenced.

The fact that seven of the eight mutations identified in XP-V cell lines result in severely truncated proteins indicates that mutations in hRAD30 are responsible for XP-V. Our results also suggest that loss of hRad30 function is not lethal. XP-V cells are hypermutable with UV light, and they are less likely than normal cells to incorporate deoxyadenosine 5′-monophosphate opposite photoproducts involving thymine (5). These observations support the notion that hRad30 functions in error-free replication of DNA containing a T-T dimer in a manner similar to the yeast Rad30 protein, and its inactivation leads to UV hypermutability and to increased incidence of sunlight-induced skin cancers in XP-V patients. Increased bypass of DNA lesions by the mutagenic DNA polymerase, Polζ, would be the cause of UV hypermutability in XP-V cells. Less drastic mutations inhRAD30 that do not elicit the clinical form of XP-V may contribute to skin cancers in the general population.

  • * To whom correspondence should be addressed. E-mail: lprakash{at}


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