Isolation and characterization of Saccharomyces cerevisiae RAD5 DNA repair gene and encoded protein.

摘要

The RAD5 gene of Saccharomyces cerevisiae is involved in the postreplication repair pathway of DNA repair. Mutants of rad5 are sensitive to ultraviolet light (UV) and other DNA damaging agents, show reduced UV induced reversion of certain ochre and frameshift alleles, and affect the stability of simple repetitive sequences within the genome. The gene has been cloned in our laboratory by complementation of the rad5-7 point mutation and the entire RAD5 gene has been sequenced. RAD5 encodes a protein of 1,169 amino acids with a calculated molecular weight of 134 kDa. RAD5 protein contains all seven conserved domains that have been identified in the superfamilies of DNA and RNA helicases, and also contains the cysteine-rich motif, a novel zinc finger domain found in several other proteins including that encoded by the DNA repair genes RAD16 and RAD18. A leucine zipper motif preceded by basic region is also present in RAD5. RAD5 is a member of a new subfamily of yeast and Drosophila proteins that are dramatically conserved over the proposed helicase domains. This group includes the DNA repair proteins RAD16 and RAD54 from yeast, and human ERCC6, as well as several transcription activators.; Deletion mutants of rad5 have been generated and examined for their effect on viability, DNA repair, UV induced mutagenesis, recombination and sporulation. The rad5 deletion mutation does not affect recombination, sporulation or viability, and UV sensitivity and UV induced mutagenesis resemble that of the point mutant. While RAD5 is shown to belong to the RAD6 epistasis group of repair genes, genetic evidence suggests that RAD5 functions primarily with RAD18 in error free post replication repair and is not involved in the overall incorporation of mutations in the genome.; RAD5 protein has been purified to near homogeneity from a RAD5 overexpressing strain of yeast and has been shown to be a single stranded DNA dependent ATPase. RAD5, however, does not show DNA helicase activity when assayed with several substrates. RAD5 may work with RAD18 in unwinding very short stretches of DNA for strand exchange during gap filling or to generate the primer for translesion DNA synthesis. RAD5 could also be a component of the replication machinery during normal and translesion DNA synthesis. Finally, RAD5 may be involved in a yeast mechanism homologous to that in humans which causes repetitive sequences to change length. This process has been implicated in many human genetic diseases including Huntington's chorea, fragile X syndrome and myotonic dystrophy, and is thought to occur through polymerase slippage events or erroneous recombination.