Structure and expression of the excision repair gene ERCC6, involved in the human disorder Cockayne's syndrome group B.

The human repair gene ERCC6--a presumed DNA (or RNA) helicase--has recently been found to function specifically in preferential nucleotide excision repair (NER). This NER subpathway is primarily directed towards repair of (the transcribed strand of) active genes. Mutations in the ERCC6 gene are responsible for the human hereditary repair disorder Cockayne's syndrome complementation group B, the most common form of the disease. In this report, the genomic organization and expression of this gene are described. It consists of at least 21 exons, together with the promoter covering a region of 82-90 kb on the genome. Postulated functional domains deduced from the predicted amino acid sequence, including 7 distinct helicase signatures, are--with one exception--encoded on separate exons. Consensus splice donor and acceptor sequences are present at all exon borders with the exception of the unusual splice donor at the end of exon VII. The 'invariable' GT dinucleotide in the consensus (C,A)AG/GTPuAGT is replaced by the exceptional GC. Based on 42 GC splice donor sequences identified by an extensive literature search we found a statistically highly significant better 'overall' match of the surrounding nucleotides to the consensus sequence compared to normal GT-sites. This confirms and extends the observation made recently by Jackson (Nucl. Acids Res., 19, 3795-3798 (1991)) derived from analysis of 26 cases. Analysis of ERCC6 cDNA clones revealed the occurrence of alternative polyadenylation, resulting in the (differential) expression of two mRNA molecules (which are barely detectable on Northern blots) of 5 and 7 kb in length.     

Overexpressed human RAD50 exhibits cell death in a p21(WAF1/CIP1)-dependent manner: its potential utility in local gene therapy of tumor.

Previously, mouse RAD50, one of the mammalian DNA recombination repair genes, was reported to have limited epitopic homology to p53. Here we report the functional characteristics of overexpressed human RAD50 (hRAD50). Transient transfection of hRAD50 in several cultured cells caused cytotoxicity. We established tetracycline-regulated, stable hRAD50 expression systems in SaOS-2 cells, which retain mutated p53, and in HeLa cells. After tetracycline withdrawal, cell death and multinucleated giant cells were observed with increased hRAD50 expression, and p21(WAF1/CIP1) but not p53 was increased. Transient transfection of hRAD50 in HCT116 p21(-/-) cells caused no cytotoxicity, but there was a significantly decreased survival rate in p21(+/+) cells. These cytotoxic effects of overexpressed hRAD50 in HeLa, SaOS-2, and HCT116 p21(+/+) cells were partially blocked by pretreatment of cells with N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a pan-caspase inhibitor. When the hRAD50 expression cDNA was injected intratumorally with liposomes, it regressed or delayed tumor development in the animal model and nitric oxide synthase expression was induced in the tumor tissues that had regressed. Our results indicate that overexpressed hRAD50 has an antiproliferation activity in vitro and in vivo in a p21-dependent manner.     

Biochemical characterization of the human RAD51 protein. I. ATP hydrolysis

The prototypical bacterial RecA protein promotes recombination/repair by catalyzing strand exchange between homologous DNAs. While the mechanism of strand exchange remains enigmatic, ATP-induced cooperativity between RecA protomers is critical for its function. A human RecA homolog, human RAD51 protein (hRAD51), facilitates eukaryotic recombination/repair, although its ability to hydrolyze ATP and/or promote strand exchange appears distinct from the bacterial RecA. We have quantitatively examined the hRAD51 ATPase. The catalytic efficiency (k(cat)/K(m)) of the hRAD51 ATPase was approximately 50-fold lower than the RecA ATPase. Altering the ratio of DNA/hRAD51 and including salts that stimulate DNA strand exchange (ammonium sulfate and spermidine) were found to affect the catalytic efficiency of hRAD51. The average site size of hRAD51 was determined to be approximately 3 nt (bp) for both single-stranded and double-stranded DNA. Importantly, hRAD51 lacks the magnitude of ATP-induced cooperativity that is a hallmark of RecA. Together, these results suggest that hRAD51 may be unable to coordinate ATP hydrolysis between neighboring protomers.     

Regulation and localization of the Bloom syndrome protein in response to DNA damage

Bloom syndrome (BS) is an autosomal recessive disorder characterized by a high incidence of cancer and genomic instability. BLM, the protein defective in BS, is a RecQ-like helicase, presumed to function in DNA replication, recombination, or repair. BLM localizes to promyelocytic leukemia protein (PML) nuclear bodies and is expressed during late S and G2. We show, in normal human cells, that the recombination/repair proteins hRAD51 and replication protein (RP)-A assembled with BLM into a fraction of PML bodies during late S/G2. Biochemical experiments suggested that BLM resides in a nuclear matrix-bound complex in which association with hRAD51 may be direct. DNA-damaging agents that cause double strand breaks and a G2 delay induced BLM by a p53- and ataxia-telangiectasia mutated independent mechanism. This induction depended on the G2 delay, because it failed to occur when G2 was prevented or bypassed. It coincided with the appearance of foci containing BLM, PML, hRAD51 and RP-A, which resembled ionizing radiation-induced foci. After radiation, foci containing BLM and PML formed at sites of single-stranded DNA and presumptive repair in normal cells, but not in cells with defective PML. Our findings suggest that BLM is part of a dynamic nuclear matrix-based complex that requires PML and functions during G2 in undamaged cells and recombinational repair after DNA damage.