Structural and functional conservation of the human homolog of the Schizosaccharomyces pombe rad2 gene, which is required for chromosome segregation and recovery from DNA damage.

Abstract

The rad2 mutant of Schizosaccharomyces pombe is sensitive to UV irradiation and deficient in the repair of UV damage. In addition, it has a very high degree of chromosome loss and/or nondisjunction. We have cloned the rad2 gene and have shown it to be a member of the Saccharomyces cerevisiae RAD2/S. pombe rad13/human XPG family. Using degenerate PCR, we have cloned the human homolog of the rad2 gene. Human cDNA has 55% amino acid sequence identity to the rad2 gene and is able to complement the UV sensitivity of the rad2 null mutant. We have thus isolated a novel human gene which is likely to be involved both in controlling the fidelity of chromosome segregation and in the repair of UV-induced DNA damage. Its involvement in two fundamental processes for maintaining chromosomal integrity suggests that it is likely to be an important component of cancer avoidance mechanisms.

Homologous regions of Fen1 and p21Cip1 compete for binding to the same site on PCNA: a potential mechanism to co-ordinate DNA replication and repair

“…We have identi®ed here an interaction between PCNA and the¯ap endonuclease Fen1 (Harrington and Lieber, 1994a,b), also known as DNase IV (Robins et al, 1994); rad2hs, (Murray et al, 1994), and MF1 (Waga et al, 1994a), using a two hybrid screen. We have veri®ed that the interaction between full length Fen1 and PCNA proteins is direct by FarWestern analysis.…”

“…One class of clones encoded p21 Cip1 , while in the second class, one clone was found to encode full length Fen1 (including a short upstream region) with eight other clones encoding fragments of Fen1 that interacted with PCNA to give signi®cant levels of b-galactosidase activity compared to controls. The minimal Fen1 fragment isolated in our screen (Figure 1) encoded the C-terminal 88 amino acids of the protein (full length 380 aa, Murray et al, 1994), thus localizing a PCNA binding site to these residues. PCNA from Drosophila melanogaster and Schizosaccharomyces pombe also interacted in this system with human Fen1 (data not shown), suggesting strong evolutionary conservation of the interaction site.…”

“…comm.). To determine if the Fen1-PCNA interaction acts through an evolutionarily conserved mechanism, we investigated the binding of the homologues of Fen1, Rad2 from S pombe (Murray et al, 1994) and RAD27 from S cerevisiae (Jacquier et al, 1992) to human PCNA. Biotinylated 20 amino acid peptides corresponding to the PCNA binding site determined for human Fen1 were synthesized for these yeast homologues ( Figure 4b).…”

“…irradiation, suggesting that RAD27 may also play a role in base excision repair (Reagan et al, 1995;Sommers et al, 1995). Mutations in S pombe rad2, which encodes a Fen1 homologue, can be complemented by expression of human Fen1 (rad2 hs ) (Murray et al, 1994). In contrast to RAD27, S pombe rad2 mutants are sensitive to u.v.…”

“…In contrast to RAD27, S pombe rad2 mutants are sensitive to u.v. irradiation, though epistasis analysis indicates that rad2 may act through an alternative pathway to the conserved excision repair pathway (Murray et al, 1994).…”