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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

Genomic Sequencing of Human and Rodent DNA Repair Gene Regions

Jane E. Lamerdin, Mishelle A. Montgomery, Stephanie A. Stilwagen, Jakob M. Kirchner, Edmund P. Salazar, Christine A. Weber, Robert S. Tebbs, Kerry W. Brookman, Larry H. Thompson, and Anthony V. Carrano
Human Genome Center, L-452, Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94550.

The ability to repair damaged DNA in cells is critical to the survival and reproduction of all eucaryotes. Deficiency of specific DNA repair processes can manifest as cancers or other diseases. The process of DNA repair occurs through two main pathways, nucleotide excision repair (NER) and mismatch repair, and each of these pathways involves the interaction of numerous proteins. Several of the NER proteins might also play a role in transcription. Three DNA repair genes, ERCC1, ERCC2, and XRCC1, have been mapped to human chromosome 19q13.2-13.3. The genomic sequence of the ERCC 1 gene has been reported previously (Martin-Gallardo et al., Nature Genetics 1:34-39, 1992). We describe here the sequencing of approximately 130 kbp of noncontiguous genomic DNA containing the mouse and human XRCC1 genes, the human and hamster ERCC2 genes, and the human ERCC4 gene (which maps to chromosome 16).

Cosmids containing the XRCC1 and ERCC2 genes were identified by hybridization with cDNA probes to the appropriate cosmid library (eg. mouse, hamster, or human chromosome 19). The human ERCC4 cosmid was identified as a secondary transformant that provided functional correction of a repair-deficient hamster cell line (UV41). Four of the five cosmids were sonicated and subcloned into M13 or phagemid vectors. The essential ERCC2 gene region in the hamster cosmid was defined and subcloned into plasmid and M13 vectors, which were subjected to exonuclease IIT to generate nested deletions. All templates were sequenced using Taq dye primer cycle sequencing kits (Applied Biosystems Division of Perkin Elmer, ABI) on an ABI 800 Catalyst Workstation. Resultant sequencing ladders were loaded on 4.75% or 6% sequencing gels and data collected on ABI 373A DNA sequencers. Sequence chromatograms were imported into GENeration (Intelligenetics, IG), where editing and assembly were performed.

Comparative genomic sequencing of the human and mouse XRCC1 gene regions reveals the presence of 26 elements that are at least 65% homologous. Seventeen of these elements correspond to the exons of XRCC1 (the human and mouse coding regions are 84% identical), and two correspond to introns whose lengths are completely conserved. The human ERCC2 gene is comprised of 23 exons and is 98% identical to the hamster gene at the protein level. Exon lengths between species are completely conserved, and some intron lengths are conserved for this gene as well. The human XRCC 1 cosmid has an average density of 1.1 Alu per kbp, but due to clustering, the local density is as high as 1.8 Alu per kbp. The human ERCC2 cosmid appears to have a similar density, and the clustering effect is even more pronounced. In one 1.4 kbp region of intron 12, there are 3.5 tandemly arrayed Alu elements, and a monomer on the opposite strand; a density of 2.8 Alu/ kbp. In contrast, the SINEs B 1 and B2 are present in the mouse XRCC 1 cosmid at a density of 0.4 per kbp, and in the hamster ERCC2 gene region at a density of 0.3 SINEs per kbp.

The human ERCC2 cosmid contains an additional 8 ORFs with excellent coding potential (as identified by XGRAIL 1.1) on the opposite strand and at the 3'-end of the ERCC2 gene. At this time, its function is unknown. The presence of a homologous region in the hamster cosmid is under investigation. The human ERCC4 gene has been recently identified and cloned by complementation analysis, but no sequence data has been reported. We have begun sequencing the cosmid containing the gene, and several putative cDNAs.

This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under contract no. W-7405-ENG48